Method of preventing or reducing the progression of prostate cancer

ABSTRACT

The embodiments include methods of preventing or reducing the progression of prostate cancer in mammals susceptible to developing prostate cancer, and to methods of reducing the incidence of clinically detected prostate cancer, using compositions containing compounds based on small peptides and a pharmaceutically acceptable carrier. The method includes, but is not limited to, administering the compounds intramuscularly, orally, intravenously, intraprostatically, intrathecally, intratumorally, intranasally, topically, transdermally, etc., either alone or conjugated to a carrier to a mammal in need thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 27, 2016, isnamed 063307-0447523_SL.txt and is 33,218 bytes in size.

BACKGROUND 1. Field of the Embodiments

The embodiments include methods of preventing or reducing theprogression of prostate cancer in mammals susceptible to developingprostate cancer, and to methods of reducing the incidence of clinicallydetected prostate cancer, using compositions containing compounds basedon small peptides and a pharmaceutically acceptable carrier. The methodsinclude, but are not limited to, administering the compositionsintramuscularly, orally, intravenously, intraperitoneally,intraprostatically, intracerebrally (intraparenchymally),intracerebroventricularly, intralesionally, intraocularly,intraarterially, intrathecally, intratumorally, intranasally, topically,transdermally, subcutaneously, or intradermally to patients in needthereof, wherein those patients who do not already have clinicallydetected prostate cancer are less likely to develop clinical prostatecancer, (i.e., the incidence of prostate cancer is reduced), and/orthose patients who have a risk of developing prostate cancer(test-detected prostate cancer), do not exhibit substantial progressionof the cancer (i.e., prevent or reduce the progression of prostatecancer).

2. Description of Related Art

The essence of many medical treatments and procedures involves theremoval or destruction of harmful or unwanted tissue. Examples of suchtreatments include the surgical removal of cancerous or pre-cancerousgrowths, the destruction of metatastic tumors through chemotherapy, andthe reduction of glandular (e.g. prostate) hyperplasia. Other examplesinclude the removal of unwanted facial hair, the removal of warts, andthe removal of unwanted fatty tissue.

There is a need for an effective composition that will destroy and henceeither facilitate the removal of or inhibit the further growth ofharmful or unwanted cells and tissue but will have mainly local effectsand minimal or absent systemic toxicity. There also is a need to reducethe need for invasive surgical intervention, even after treatment withan effective composition.

Some agents known to have the ability to destroy and hence eitherfacilitate the removal of or inhibit the further growth of harmful orunwanted cells and tissue are disclosed in U.S. patent application Ser.No. 14/808,713, filed Jul. 24, 2015, entitled: METHODS OF REDUCING THENEED FOR SURGERY IN PATIENTS SUFFERING FROM BENIGN PROSTATICHYPERPLASIA; U.S. patent application Ser. No. 14/606,683, filed Jan. 27,2015, entitled: METHOD OF TREATING DISORDERS REQUIRING DESTRUCTION ORREMOVAL OF CELLS, U.S. application Ser. No. 14/738,551, filed Jun. 12,2015, entitled: COMBINATION COMPOSITIONS FOR TREATING DISORDERSREQUIRING REMOVAL OR DESTRUCTION OF UNWANTED CELLULAR PROLIFERATIONS,U.S. patent application Publication Nos. 2007/0237780 (now abandoned);2003/0054990 (now U.S. Pat. No. 7,172,893); 2003/0096350 (now U.S. Pat.No. 6,924,266); 2003/0096756 (now U.S. Pat. No. 7,192,929); 2003/0109437(now U.S. Pat. No. 7,241,738); 2003/0166569 (now U.S. Pat. No.7,317,077); 2005/0032704 (now U.S. Pat. No. 7,408,021); and 2015/0148303(now U.S. Pat. No. 9,243,035), the disclosures of each of which areincorporated by reference herein in their entirety.

Cancer is an abnormality in a cell's internal regulatory mechanisms thatresults in uncontrolled growth and reproduction of the cell. Normalcells make up tissues, and when these cells lose their ability to behaveas a specified, controlled, and coordinated unit, (dedifferentiation),the defect leads to disarray amongst the cell population. When thisoccurs, a tumor is formed.

Benign overgrowths of tissue are abnormalities in which it is desirableto remove cells from an organism. Benign tumors are cellularproliferations that do not metastasize throughout the body but do,however, cause disease symptoms. Such tumors can be lethal if they arelocated in inaccessible areas in organs such as the brain. There arebenign tumors of organs including lung, brain, skin, pituitary, thyroid,adrenal cortex and medulla, ovary, uterus, testis, connective tissue,muscle, intestines, ear, nose, throat, tonsils, mouth, liver, gallbladder, pancreas, prostate, heart, and other organs.

Surgery often is the first step in the treatment of cancer. Theobjective of surgery varies. Sometimes it is used to remove as much ofthe evident tumor as possible, or at least to “debulk” it (remove themajor bulk(s) of tumor so that there is less that needs to be treated byother means). Depending on the cancer type and location, surgery mayalso provide some symptomatic relief to the patient. For instance, if asurgeon can remove a large portion of an expanding brain tumor, thepressure inside the skull will decrease, leading to improvement in thepatient's symptoms.

Not all tumors are amenable to surgery. Some may be located in parts ofthe body that make them impossible to completely remove. Examples ofthese would be tumors in the brainstem (a part of the brain thatcontrols breathing) or a tumor which has grown in and around a majorblood vessel. In these cases, the role of surgery is limited due to thehigh risk associated with tumor removal.

In some cases, surgery is not used to debulk tumor tissue because it issimply not necessary. An example is Hodgkin's lymphoma, a cancer of thelymph nodes that responds very well to combinations of chemotherapy andradiation therapy. In Hodgkin's lymphoma, surgery is rarely needed toachieve cure, but almost always used to establish a diagnosis.

Chemotherapy is another common form of cancer treatment. Essentially, itinvolves the use of medications (usually given by mouth or injection)which specifically attack rapidly dividing cells (such as those found ina tumor) throughout the body. This makes chemotherapy useful in treatingcancers that have already metastasized, as well as tumors that have ahigh chance of spreading through the blood and lymphatic systems but arenot evident beyond the primary tumor. Chemotherapy may also be used toenhance the response of localized tumors to surgery and radiationtherapy. This is the case, for example, for some cancers of the head andneck.

Unfortunately, other cells in the human body that also normally dividerapidly (such as the lining of the stomach and hair) also are affectedby chemotherapy. For this reason, many chemotherapy agents induceundesirable side effects such as nausea, vomiting, anemia, hair loss orother symptoms. These side effects are temporary, and there existmedications that can help alleviate many of these side effects. As ourknowledge has continued to grow, researchers have devised newerchemotherapeutic agents that are not only better at killing cancercells, but that also have fewer side effects for the patient.

Chemotherapy is administered to patients in a variety of ways. Someinclude pills and others are administered by an intravenous or otherinjection. For injectable chemotherapy, a patient goes to the doctor'soffice or hospital for treatment. Other chemotherapeutic agents requirecontinuous infusion into the bloodstream, 24 hours a day. For thesetypes of chemotherapy, a minor surgical procedure is performed toimplant a small pump worn by the patient. The pump then slowlyadministers the medication. In many cases, a permanent port is placed ina patient's vein to eliminate the requirement of repeated needle sticks.

Benign tumors and malformations also can be treated by a variety ofmethods including surgery, radiotherapy, drug therapy, thermal orelectric ablation, cryotherapy, and others. Although benign tumors donot metastasize, they can grow large and they can recur. Surgicalextirpation of benign tumors has all the difficulties and side effectsof surgery in general and oftentimes must be repeatedly performed forsome benign tumors, such as for pituitary adenomas, meningeomas of thebrain, prostatic hyperplasia, and others. In addition, some patients whoreceive non-surgical treatment to ameliorate the symptoms caused bybenign tumors, still require subsequent invasive surgical intervention.Lepor, “Medical Treatment of Benign Prostatic Hyperplasia,” Reviews inUrology, Vol. 13, No. 1, pp. 20-33 (2011), discloses a variety ofstudies of the efficacy of drug therapies in treating BPH, and the needfor subsequent invasive surgical treatment.

The role of androgens in the development of benign prostatic hyperplasiain men is well documented (Wilson, N. Engl. J. Med. 317: 628-629, 1987).In fact, benign prostatic hyperplasia does not develop in the absence ofthe testes (referred to in Wendel et al., J. Urol. 108: 116-119, 1972).

Blockade of testicular androgen secretion by surgical or medical (LHRHagonist) castration is known to decrease prostatic size (Auclair et al.,Biochem. Biophys. Res. Commun. 76: 855-862, 1977; Auclair et al.,Endocrinology 101: 1890-1893, 1977; Labrie et al., Int. J. Andrology,suppl. 2 (V. Hansson, ed.), Scriptor Publisher APR, pp. 303-318, 1978;Labrie et al., J. Andrology 1: 209-228, 1980; Tremblay and Belanger,Contraception 30: 483-497, 1984; Tremblay et al., Contraception 30:585-598, 1984; Dube et al., Acta Endocrinol. (Copenh) 116: 413-417,1987; Lacoste et al., Mol. Cell. Endocrinol. 56: 141-147, 1988; White,Ann. Surg. 22: 1-80, 1895; Faure et al., Fertil. Steril. 37: 416-424,1982; Labrie et al., Endocrine Reviews 7: 67-74, 1986; Huggins andStevens, J. Urol. 43: 705-714, 1940; Wendel et al., J. Urol. 108:116-119, 1972; Peters and Walsh, N. Engl. J. Med. 317: 599-604, 1987;Gabrilove et al., J. Clin. Endocrinol. Metab. 64: 1331-1333, 1987).

Several studies have shown that treatment with an antiandrogen alsodecreases prostatic size (Neri et al., Endocrinology, 82: 311-317, 1968;Neri et al., Investigative Urology, 10: 123-130, 1972; Tunn et al., ActaEndocrinol. (Copenh.) 91: 373-384, 1979; Seguin et al., Mol. Cell.Endocrinol., 21: 37-41, 1981; Lefebvre et al., The Prostate 3: 569-578,1982; Marchetti and Labrie, J. Steroid Biochem, 29: 691-698, 1988;Lacoste et al., Mol. Cell. Endocrinol. 56: 141-147, 1988; Tunn et al.,Invest. Urol. 18: 289-292, 1980; Scott and Wade, J. Urol. 101: 81-85,1969; Caine et al., J. Urol. 114: 564-568, 1975; Stone et al., J. Urol.141: 240A, 1989; Clejan et al., J. Urol. 141: 534A, 1989).

U.S. Pat. No. 3,423,507 discloses the use of the antiandrogencyproterone acetate (1α,2β-methylene-6-chloro-17α-acetoxy-6-dehydroprogesterone) for thetreatment of benign prostatic hyperplasia. Pure antiandrogens (U.S. Pat.No. 4,329,364) cause an increase in testosterone secretion, which canresult in a higher degree of aromatization into estrogens, a situationexpected from current knowledge to have negative effects on prostatichyperplasia (Jacobi et al., Endocrinology 102: 1748-1755, 1978). Severalstudies have shown that treatment with the combination of chemicalcastration (LHRH agonist) and an antiandrogen cause greater inhibitionof prostatic size than either treatment used alone (Seguin et al., Mol.Cell. Endocrinol. 21: 37-41, 1981; Lefebvre et al., The Prostate 3:569-578, 1982; Marchetti and Labrie, J. Steroid Biochem. 29: 691-698,1988.

In the prostate as well as in many other tissues, testosterone isirreversibly converted by 5α-reductase into the more potent androgendihydrotestosterone (Bruchovsky and Wilson, J. Biol. Chem. 243:2012-2021, 1968; Wilson, Handbook of Physiology 5 (section 7), pp.491-508, 1975). Inhibitors of 5 α-reductase have been found to inhibitprostatic growth (Brooks et al., Endocrinology 109: 830, 1981; Brooks etal., Proc. Soc. Exp. Biol. Med. 169: 67, 1982; Brooks et al., Prostate3: 35, 1982; Wenderoth et al., Endocrinology 113, 569-573, 1983;McConnell et al., J. Urol. 141: 239A, 1989); Stoner, E., Lecture on therole of 5.alpha.-reductase inhibitor in benign prostatic hypertropy,84th AUA Annual Meeting, Dallas, May 8, 1989.

The inhibitory effect of the 5 α-reductase inhibitor Merck L 652,931 onprostatic and seminal vesicle development in the prepubertal rat wasdescribed in Proc. 71st Annual Meeting of Endocr. Soc. abst. #1165, p.314, 1989. The inhibitory effect of MK-906 on dihydrotestosteroneformation in men has been described in men by Gormley et al., in Proc.71st Annual Meeting of Endocr. Soc., abst. #1225, p. 329, 1989;Imperato-McGinley et al., in Proc. 71st Annual Meeting of Endocr. Soc.,abst. #1639, p. 432, 1989; Geller and Franson, in Proc. 71st AnnualMeeting of Endocr. Soc., abst. #1640, p. 432, 1989 and Tenover et al.,in Proc. 71st Annual Meeting of Endocr. Soc., abst. #583, p. 169, 1989.The activity of the 5 α-reductase inhibitorsN,N-diethyl-4-methyl-3-oxo-4-aza-5.alpha.-androstane-17.beta.-carboxamide(4-MA) and 6-methylene-4-pregnene-3,20-dione (LY 207320) has beendescribed by Toomey et al., Proc. 71st Annual Meeting of Endocr. Soc.,abst. #1226, p. 329, 1989.

In addition to the well-known effect of androgens on prostatic growth,there are many studies which show that estrogens play also a role inproliferation of the prostate (Walsh and Wilson, J. Clin. Invest. 57:1093-1097, 1976; Robinette et al., Invest. Urol. 15: 425-432, 1978;Moore et al., J. Clin. Invest. 63: 351-257, 1979). Moreover, estrogenshave been shown to enhance androgen-induced prostatic growth in the dog(Walsh and Wilson, J. Clin. Invest. 57: 1093-1097, 1976; Jacobi et al.,Endocrinology 102: 1748-1755, 1978; Tunn et al., Urol. Int. 35: 125-140,1980). A possible explanation of this enhancing effect of estrogen onandrogen-induced prostate growth, is the observation that 17β-estradiolhas been shown to increase androgen binding in the dog prostate (Mooreet al., J. Clin. Invest. 63: 351-357, 1979).

The antiestrogen Tamoxifen has been shown to improve steroid-inducedbenign prostatic hyperplasia in the dog (Funke et al., Acta Endocrinol.100: 462-472, 1982). Administration of the antiestrogen Tamoxifen inassociation with the steroidal antiandrogen cyproterone acetate inpatients suffering from benign prostatic hyperplasia showed beneficialeffects on the symptoms of the disease (Di Silverio et al., inIpertrofia Prostatica Benigna (F. Di Silverio, F. Neumann and M.Tannenbaum, eds), Excerpta Medica, pp. 117-125, 1986). In U.S. Pat. No.4,310,523, it is proposed that a combination of an antiandrogen and anantiestrogen is effective for the prophylaxis and/or therapy of benignprostatic hyperplasia. Tamoxifen, however, has intrinsic estrogenicactivity which limits its effectiveness.

Estrogen formation resulting from aromatization of androgens, occurs atseveral sites. In the male, aromatization of androgens has beendemonstrated in the testis, adipose and muscle tissue, skin, liver,brain and prostate (Schweikert et al., J. Clin. Endocrinol. Metab. 40:413-417, 1975; Folker and James, J. Steroid Biochem. 49: 687-690, 1983;Longcope et al., J. Clin. Endocrinol. Metab. 46: 146-152, 1978; Lacosteand Labrie, unpublished data; Stone et al., The Prostate 9: 311-318,1986; Stone et al., Urol. Res. 15: 165-167, 1987). There is evidence foran increased production of estrogens in the prostatic tissue of benignprostatic hyperplasia patients (Stone et al., The Prostate 9: 311-318,1986). Such data indicate that the local formation of estrogens may playa crucial role in stimulating prostatic growth in excess of the actionpredicted by circulating estrogens.

U.S. Pat. No. 4,472,382 discloses treatment of BPH with an antiandrogenand certain peptides which act as LH-RH agonists. U.S. Pat. No.4,596,797 discloses aromatase inhibitors as a method of prophylaxisand/or treatment of prostatic hyperplasia. U.S. Pat. No. 4,760,053describes a treatment of certain cancers which combines an LHRH agonistwith an antiandrogen and/or an antiestrogen and/or at least oneinhibitor of sex steroid biosynthesis. U.S. Pat. No. 4,775,660 disclosesa method of treating breast cancer with a combination therapy which mayinclude surgical or chemical prevention of ovarian secretions andadministering an antiandrogen and an antiestrogen.

U.S. Pat. No. 4,659,695 discloses a method of treatment of prostatecancer in susceptible male animals including humans whose testicularhormonal secretions are blocked by surgical or chemical means, e.g. byuse of an LHRH agonist, which comprises administering an antiandrogen,e.g. flutamide, in association with at least one inhibitor of sexsteroid biosynthesis, e.g. aminoglutethimide and/or ketoconazole. Thedisclosures of each of the above-mentioned patents (U.S. Pat. Nos.4,472,382, 4,596,797, 4,760,053, 4,775,660, and 4,659,695) areincorporated by reference herein in their entireties.

BPH is caused by increased activity of both androgens and estrogens.Because of such a dual etiology of BPH, proposed hormonal therapies havebeen less than satisfactory and have all been unpredictable while,frequently, causing unacceptable side-effects. Moreover, the prior arttreatment seldomly resulted in a decrease in prostatic volume aboveabout 20 to 30% with inconsistent effects on the symptomatology (Scottand Wade, J. Urol. 101: 81-85, 1969; Caine et al., J. Urol. 114:564-568, 1975; Peters and Walsh, New Engl. J. Med. 317: 599-604, 1987;Gabrilove et al., J. Clin. Endocrinol. Metab. 64: 1331-1333, 1987; Stoneet al., J. Urol. 141: 240A, 1989; Clejan et al., J. Urol. 141: 534A,1989; Stoner, E., Lecture on the role of 5 α-reductase inhibitor inbenign prostatic hypertrophy, 84th AUA Annual Meeting, Dallas, May 8,1989.

The elucidation of the mechanism summarized above has resulted in therecent development of effective agents to control, and in many casesreverse, the advance of BPH. In the forefront of these agents is Merck &Co., Inc.s' product PROSCAR® (finasteride). The effect of this compoundis to inhibit the enzyme testosterone 5α reductase, which convertstestosterone into 5α-dihydrotesterone, resulting in a reduced rate ofprostatic enlargement, and often reduction in prostatic mass.

Prostate cancer is known as a disease with an extremely high prevalencerelative to its clinical incidence in the population. Prostate cancerhas a high asymptomatic incidence and a long asymptomatic duration.Prostate cancer has an interval of 7 to 14 years on average during whichthe cancer is present but is preclinical because it is not detected bytypical clinical or laboratory examinations (see Etzioni, R et al., Am JEpidemiol. Vol. 148, pp. 775-85 (1998); and Gulati, R, et al., CancerEpidemiol Biomarkers Prev; Vol. 20(5), pp. 740-50 (2011)). Thesepreclinical asymptomatic prostate cancers prior to their overt diagnosisare a reasonable target for treatment which can benefit patients beforethe cancers have become clinically apparent or detected otherwise.

In all or most of these instances, there is a need for treatments thatcan remove or destroy the unwanted cellular elements without the risksand side effects of conventional therapies, or treatments that canremove the unwanted cellular elements with more precision. There also isa need to develop therapies that can prevent or reduce the progressionof prostate cancer, or reduce the incidence of clinically detectedprostate cancer.

Throughout this description, including the foregoing description ofrelated art, any and all publicly available documents described herein,including any and all U.S. patent published patent applications, arespecifically incorporated by reference herein in their entirety. Theforegoing description of related art is not intended in any way as anadmission that any of the documents described therein, including pendingU.S. patent applications, are prior art to the present disclosure.Moreover, the description herein of any disadvantages associated withthe described products, methods, and/or apparatus, is not intended tolimit the embodiments. Indeed, aspects of the embodiments may includecertain features of the described products, methods, and/or apparatuswithout suffering from their described disadvantages.

SUMMARY OF THE EMBODIMENTS

There remains a need in the art for new, less toxic, and less frequent(e.g., avoiding the need to take medications daily or weekly) treatmentsfor preventing or reducing the progression or incidence of prostatecancer. There also remains a need in the art for such treatments thatreduce the incidence of clinically detected prostate cancer. Theembodiments satisfy these needs.

This disclosure is premised in part on the discovery that certain NTPpeptides, including a specific peptide described by the amino acidsequenceIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu (SEQID NO: 66), are capable of preventing or reduce the progression ofprostate cancer, as well as reducing the incidence of clinicallydetected prostate cancer.

This disclosure also is premised in part on the discovery that certainNTP peptides, including a specific peptide described by the amino acidsequenceIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu (SEQID NO: 66), either alone or in combination with an additional activeagent capable of treating and/or killing unwanted cellularproliferations in mammals, provide an unexpected improvement in patientssuffering from or susceptible to developing prostate cancer.

Some embodiments are directed to methods of preventing or reducing theprogression of prostate cancer in mammals comprising administering to amammal in need thereof a therapeutically effective amount of acomposition comprising an NTP peptide, either alone or in combinationwith at least one additional active agent capable of treating and/orkilling unwanted cellular proliferations in mammals. In an embodiment,there are provided methods of reducing the incidence of prostate cancerin mammals comprising administering to a mammal in need thereof atherapeutically effective amount of a composition comprising an NTPpeptide, either alone or in combination with at least one additionalactive agent capable of treating and/or killing unwanted cellularproliferations in mammals.

The compositions can be administered intramuscularly, orally,intravenously, intraperitoneally, intracerebrally (intraparenchymally),intracerebroventricularly, intratumorally, intralesionally,intradermally, intrathecally, intranasally, intraocularly,intraarterially, topically, transdermally, via an aerosol, infusion,bolus injection, implantation device, sustained release system etc.Alternatively, the NTP peptides can be expressed in vivo byadministering a gene that expresses the NTP peptides, by administering avaccine that induces such production or by introducing cells, bacteriaor viruses that express the peptide in vivo, because of geneticmodification or otherwise.

In another embodiment, administering a composition comprising an NTPpeptide, either alone or in combination with at least one additionalactive agent capable of treating and/or killing unwanted cellularproliferations in mammals reduces the incidence of clinically detectedprostate cancer by more than 15%, when compared to the known expectedincidence of clinically detected prostate cancer.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory and are intended to providefurther explanation of the embodiments as claimed. Other objects,advantages, and features will be readily apparent to those skilled inthe art from the following detailed description of the embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present proteins, nucleotide sequences, peptides,compositions, active agents, etc., and methods are described, it isunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, vectors, and reagents described, asthese may vary. It also is to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present embodiments which willbe limited only by the appended claims.

Terms and phrases used herein are defined as set forth below unlessotherwise specified. Throughout this description, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, a reference to “a host cell”includes a plurality of such host cells, and a reference to “anantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Amino acids and amino acid residues described herein may be referred toaccording to the accepted one or three-letter code provided in the tablebelow.

TABLE 1 Three-Letter Amino One-Letter Acid Symbol Symbol Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

The expression “NTP peptide” refers to peptides comprising amino acidsequences corresponding to at least a part of the amino acid sequence ofNeural Thread Proteins or to fragments of Neural Thread Proteins andincludes homologues, derivatives, variants, fusion proteins, and peptidemimetics of such peptides unless the context indicates otherwise. Theexpression “NTP peptide” also refers to a peptide or other compositionof matter claimed in one or more of the following U.S. patentapplication Publication Nos. 2007/0237780 (now abandoned); 2003/0054990(now U.S. Pat. No. 7,172,893); 2003/0096350 (now U.S. Pat. No.6,924,266); 2003/0096756 (now U.S. Pat. No. 7,192,929); 2003/0109437(now U.S. Pat. No. 7,241,738); 2003/0166569 (now U.S. Pat. No.7,317,077); and 2005/0032704 (now U.S. Pat. No. 7,408,021). Thedisclosures of each of these applications are incorporated by referenceherein in their entirety. Specific peptides are listed below.

1) SEQ ID NO. 1: MEFSLLLPRLECNGA  orMet-Glu-Phe-Ser-Leu-Leu-Leu-Pro-Arg-Leu-Glu-Cys- Asn-Gly-Ala 2) SEQ ID NO. 2: GAISAHRNLRLPGSS  orGly-Ala-Ile-Ser-Ala-His-Arg-Asn-Leu-Arg-Leu-Pro- Gly-Ser-Ser 3) SEQ ID NO. 3: DSPASASPVAGITGMCT  orAsp-Ser-Pro-Ala-Ser-Ala-Ser-Pro-Val-Ala-Gly-Ile- Thr-Gly-Met-Cys-Thr 4) SEQ ID NO. 4: MCTHARLILYFFLVEM  orMet-Cys-Thr-His-Ala-Arg-Leu-Ile-Leu-Tyr-Phe-Phe- Leu-Val-Glu-Met 5) SEQ ID NO. 5: YFFLVEMEFLH  orTyr-Phe-Phe-Leu-Val-Glu-Met-Glu-Phe-Leu-His  6) SEQ ID NO. 6:VGQAGLELPTS  or Val-Gly-Gln-Ala-Gly-Leu-Glu-Leu-Pro-Thr-Ser 7) SEQ ID NO. 7: DDPSVSASQSARYRTGH  orAsp-Asp-Pro-Ser-Val-Ser-Ala-Ser-Gln-Ser-Ala- Arg-Tyr-Arg-Thr-Gly-His 8) SEQ ID NO. 8: TGHHARLCLANFCG  orThr-Gly-His-His-Ala-Arg-Leu-Cys-Leu-Ala-Asn- Phe-Cys-Gly 9) SEQ ID NO. 9: ANFCGRNRVSLMCPSWS  orAla-Asn-Phe-Cys-Gly-Arg-Asn-Arg-Val-Ser-Leu- Met-Cys-Pro-Ser-Trp-Ser 10) SEQ ID NO. 10: PELKQSTCLSLPKCWDYRR  orPro-Glu-Leu-Lys-Gln-Ser-Thr-Cys-Leu-Ser-Leu-Pro-Lys-Cys-Trp-Asp-Tyr-Arg-Arg  11) SEQ ID NO. 11: LKQSTCLSLPKCWDYRR or Leu-Lys-Gln-Ser-Thr-Cys-Leu-Ser-Leu-Pro-Lys- Cys-Trp-Asp-Tyr-Arg-Arg 12) SEQ ID NO. 12: STCLSLPKCWDYRR  orSer-Thr-Cys-Leu-Ser-Leu-Pro-Lys-Cys-Trp-Asp- Tyr-Arg-Arg 13) SEQ ID NO. 13: LSLPKCWDYRR  orLeu-Ser-Leu-Pro-Lys-Cys-Trp-Asp-Tyr-Arg-Arg  14) SEQ ID NO. 14:KCWDYRRAAVPGL  or Lys-Cys-Trp-Asp-Tyr-Arg-Arg-Ala-Ala-Val-Pro-Gly- Leu 15) SEQ ID NO. 15: KCWDYRRAAVPGLFILFFL  orLys-Cys-Trp-Asp-Tyr-Arg-Arg-Ala-Ala-Val-Pro-Gly-Leu-Phe-Ile-Leu-Phe-Phe-Leu  16) SEQ ID NO. 16:KCWDYRRAAVPGLFILFFLRHRCP  orLys-Cys-Trp-Asp-Tyr-Arg-Arg-Ala-Ala-Val-Pro-Gly-Leu-Phe-Ile-Leu-Phe-Phe-Leu-Arg-His-Arg-Cys-Pro  17) SEQ ID NO. 17: KCWDYRRAAVPGLFILFFLRHRCPTLTQDEVQWCDHSS or Lys-Cys-Trp-Asp-Tyr-Arg-Arg-Ala-Ala-Val-Pro-Gly-Leu-Phe-Ile-Leu-Phe-Phe-Leu-Arg-His-Arg-Cys-Pro-Thr-Leu-Thr-Gln-Asp-Glu-Val-Gln-Trp-Cys-Asp-His- Ser-Ser 18) SEQ ID NO. 18: WDYRR  or Trp-Asp-Tyr-Arg-Arg  19) SEQ ID NO. 19:FILFFLRHRCPTL  or Phe-Ile-Leu-Phe-Phe-Leu-Arg-His-Arg-Cys-Pro-Thr- Leu 20) SEQ ID NO. 20: FILFFLRHRCPTLTQDEVQWCDHSS  orPhe-Ile-Leu-Phe-Phe-Leu-Arg-His-Arg-Cys-Pro-Thr-Leu-Thr-Gln-Asp-Glu-Val-Gln-Trp-Cys-Asp-His-Ser- Ser  21) SEQ ID NO. 21:HRCPTLTQDEVQWCDHSSLQPSTPEIKHP  orHis-Arg-Cys-Pro-Thr-Leu-Thr-Gln-Asp-Glu-Val-Gln-Trp-Cys-Asp-His-Ser-Ser-Leu-Gln-Pro-Ser-Thr-Pro- Glu-Ile-Lys-His-Pro 22) SEQ ID NO. 22: PASASQVAGTKDMH  orPro-Ala-Ser-Ala-Ser-Gln-Val-Ala-Gly-Thr-Lys-Asp- Met-His 23) SEQ ID NO. 23: DMHHYTWLIFIFIFNFLR  orAsp-Met-His-His-Tyr-Thr-Trp-Leu-Ile-Phe-Ile-Phe-Ile-Phe-Asn-Phe-Leu-Arg  24) SEQ ID NO. 24: HYTWLIFIFIFNFLRQSLN  orHis-Tyr-Thr-Trp-Leu-ILe-Phe-Ile-Phe-Ile-Phe-Asn-Phe-Leu-Arg-Gln-Ser-Leu-Asn  25) SEQ ID NO. 25: SVTQAGVQWRNLGSLQPLPPGFKLFSCPSLLSSWDYRRPPRLANF  orSer-Val-Thr-Gln-Ala-Gly-Val-Gln-Trp-Arg-Asn-Leu- Gly-Ser-Leu-Gln-Pro-Leu-Pro-Pro-Gly-Phe-Lys-Leu-Phe-Ser-Cys-Pro-Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg-Pro-Pro-Arg-Leu-Ala-Asn-Phe  26) SEQ ID NO. 26:PGFKLFSCPSLLSSWDYRR  or Pro-Gly-Phe-Lys-Leu-Phe-Ser-Cys-Pro-Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg  27) SEQ ID NO. 27:FKLFSCPSLLSSWDYRRPPRLANF  orPhe-Lys-Leu-Phe-Ser-Cys-Pro-Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg-Pro-Pro-Arg-Leu-Ala-Asn-Phe  28) SEQ ID NO. 28:FSCPSLLSSWDYRR  or Phe-Ser-Cys-Pro-Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg  29) SEQ ID NO. 29: SLLSSWDYRR  orSer-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg  30) SEQ ID NO. 30: SSWDY  orSer-Ser-Trp-Asp-Tyr  31) SEQ ID NO. 31: SSWDYRR  orSer-Ser-Trp-Asp-Tyr-Arg-Arg  32) SEQ ID NO. 32:SSWDYRRPPRLANFFVFLVEMGFTM  or Ser-Ser-Trp-Asp-Tyr-Arg-Arg-Pro-Pro-Arg-Leu-Ala-Asn-Phe-Phe-Val-Phe-Leu-Val-Glu-Met-Gly-Phe-Thr- Met  33) SEQ ID NO. 33:FVFLVEMGFTM  or Phe-Val-Phe-Leu-Val-Glu-Met-Gly-Phe-Thr-Met 34) SEQ ID NO. 34: MGFTMFARLILISGPCDLPASAS  orMet-Gly-Phe-Thr-Met-Phe-Ala-Arg-Leu-Ile-Leu-Ile-Ser-Gly-Pro-Cys-Asp-Leu-Pro-Ala-Ser-Ala-Ser  35) SEQ ID NO. 35: ISGPC or Ile-Ser-Gly-Pro-Cys  36) SEQ ID NO. 36: DLPASASQSAGITGVSH  orAsp-Leu-Pro-Ala-Ser-Ala-Ser-Gln-Ser-Ala-Gly-Ile- Thr-Gly-Val-Ser-His 37) SEQ ID NO. 37: GVSHHARLIFNFCLFEM  orGly-Val-Ser-His-His-Ala-Arg-Leu-Ile-Phe-Asn-Phe- Cys-Leu-Phe-Glu-Met 38) SEQ ID NO. 38: NFCLFEMESH  orAsn-Phe-Cys-Leu-Phe-Glu-Met-Glu-Ser-His  39) SEQ ID NO. 39: SVTQAGVQWPNLGSLQPLPPGLKRFSCLSLPSSWDYGHLPPHPANF orSer-Val-Thr-Gln-Ala-Gly-Val-Gln-Trp-Pro-Asn-Leu-Gly-Ser-Leu-Gln-Pro-Leu-Pro-Pro-Gly-Leu-Lys-Arg-Phe-Ser-Cys-Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr-Gly-His-Leu-Pro-Pro-His-Pro-Ala-Asn-Phe  40) SEQ ID NO. 40:PPGLKRFSCLSLPSSWDYG  or Pro-Pro-Gly-Leu-Lys-Arg-Phe-Ser-Cys-Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr-Gly  41) SEQ ID NO. 41: FSCLSLPSSWDYGH  orPhe-Ser-Cys-Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr- Gly-His 42) SEQ ID NO. 42: LSLPSSWDY  or Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr 43) SEQ ID NO. 43:  SSWDYGHLPPHPANFCIFIRGGVSPYLSGWSQTPDLR or Ser-Ser-Trp-Asp-Tyr-Gly-His-Leu-Pro-Pro-His-Pro-Ala-Asn-Phe-Cys-Ile-Phe-Ile-Arg-Gly-Gly-Val-Ser-Pro-Tyr-Leu-Ser-Gly-Trp-Ser-Gln-Thr-Pro-Asp-Leu- Arg  44) SEQ ID NO. 44:PGFFKLFSCPSLLSSWDYRR  orPro-Gly-Phe-Phe-Lys-Leu-Phe-Ser-Cys-Pro-Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg  45) SEQ ID NO. 45: PELKQSTCLSLPKCWDYRR or Pro-Glu-Leu-Lys-Gln-Ser-Thr-Cys-Leu-Ser-Leu-Pro-Lys-Cys-Trp-Asp-Tyr-Arg-Arg  46) SEQ ID NO. 46: PPGLKRFSCLSLPSSWDYG  orPro-Pro-Gly-Leu-Lys-Arg-Phe-Ser-Cys-Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr-Gly  47) SEQ ID NO. 47: FSCLSLPSSWDYGH  orPhe-Ser-Cys-Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr- Gly-His 48) SEQ ID NO. 48: STCLSLPKCWDYRR  orSer-Thr-Cys-Leu-Ser-Leu-Pro-Lys-Cys-Trp-Asp-Tyr- Arg-Arg 49) SEQ ID NO. 49: FSCPSLLSSWDYRR  orPhe-Ser-Cys-Pro-Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr- Arg-Arg 50) SEQ ID NO. 50: LSLPSSWDY  or Leu-Ser-Leu-Pro-Ser-Ser-Trp-Asp-Tyr 51) SEQ ID NO. 51: LSLPKCWDYRR  orLeu-Ser-Leu-Pro-Lys-Cys-Trp-Asp-Tyr-Arg-Arg  52) SEQ ID NO. 52:SLLSSWDYRR or Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg 53) SEQ ID NO. 53: LPSSWDYRR or Leu-Pro-Ser-Ser-Trp-Asp-Tyr-Arg-Arg 54) SEQ ID NO. 54: SSWDYRR or Ser-Ser-Trp-Asp-Tyr-Arg-Arg 55) SEQ ID NO. 55: SSWDY or Ser-Ser-Trp-Asp-Tyr  56) SEQ ID NO. 56:SSWDYRRFILFFL or Ser-Ser-Trp-Asp-Tyr-Arg-Arg-Phe-Ile-Leu-Phe-Phe- Leu 57) SEQ ID NO. 57: WDYRRFIFNFL orTrp-Asp-Tyr-Arg-Arg-Phe-Ile-Phe-Asn-Phe-Leu  58) SEQ ID NO. 58: FNFCLFor Phe-Asn-Phe-Cys-Leu-Phe  59) SEQ ID NO. 59: FIFNFL orPhe-Ile-Phe-Asn-Phe-Leu  60) SEQ ID NO. 60: PASASPVAGITGM orPro-Ala-Ser-Ala-Ser-Pro-Val-Ala-Gly-Ile-Thr-Gly- Met  61) SEQ ID NO. 61:PASASQVAGTKDM or Pro-Ala-Ser-Ala-Ser-Gln-Val-Ala-Gly-Thr-Lys-Asp- Met 62) SEQ ID NO. 62: PASASQSAGITGV orPro-Ala-Ser-Ala-Ser-Gln-Ser-Ala-Gly-Ile-Thr-Gly- Val  63) SEQ ID NO. 63:PASASPVAG or Pro-Ala-Ser-Ala-Ser-Pro-Val-Ala-Gly  64) SEQ ID NO. 64:FFLVEM or Phe-Phe-Leu-Val-Glu-Met  65) SEQ ID NO. 65: SVTQAGVQW orSer-Val-Thr-Gln-Ala-Gly-Val-Gln-Trp  66) SEQ ID NO. 66:IDQQVLSRIKLEIKRCL or Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu  67) SEQ ID NO. 67: LSRIKLEIK orLeu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys  68) SEQ ID NO. 68:GDHGRPNLSRLKLAIKYEVKKM orGly-Asp-His-Gly-Arg-Pro-Asn-Leu-Ser-Arg-Leu-Lys-Leu-Ala-Ile-Lys-Tyr-Glu-Val-Lys-Lys-Met  69) SEQ ID NO. 69:QQSIAVKFLAVFGVSI or Gln-Gln-Ser-Ile-Ala-Val-Lys-Phe-Leu-Ala-Val-Phe-Gly-Val-Ser-Ile  70) SEQ ID NO. 70: GLLFPVFSVCYLIAPKSPLGL orGly-Leu-Leu-Phe-Pro-Val-Phe-Ser-Val-Cys-Tyr-Leu-Ile-Ala-Pro-Lys-Ser-Pro-Leu-Gly-Leu  71) SEQ ID NO. 71: MMVCWNRFGKWVYFIor Met-Met-Val-Cys-Trp-Asn-Arg-Phe-Gly-Lys-Trp-Val- Tyr-Phe-Ile 72) SEQ ID NO. 72: SAIFNFGPRYLYHGV orSer-Ala-Ile-Phe-Asn-Phe-Gly-Pro-Arg-Tyr-Leu-Tyr- His-Gly-Val 73) SEQ ID NO. 73: PFYFLILVRIISFLI orPro-Phe-Tyr-Phe-Leu-Ile-Leu-Val-Arg-Ile-Ile-Ser- Phe-Leu-Ile 74) SEQ ID NO. 74: GDMEDVLLNCTLLKR orGly-Asp-Met-Glu-Asp-Val-Leu-Leu-Asn-Cys-Thr-Leu- Leu-Lys-Arg 75) SEQ ID NO. 75: SSRFRFWGALVCSMD orSer-Ser-Arg-Phe-Arg-Phe-Trp-Gly-Ala-Leu-Val-Cys- Ser-Met-Asp 76) SEQ ID NO. 76: SCRFSRVAVTYRFIT orSer-Cys-Arg-Phe-Ser-Arg-Val-Ala-Val-Thr-Tyr-Arg- Phe-Ile-Thr 77) SEQ ID NO. 77: LLNIPSPAVWMARNT orLeu-Leu-Asn-Ile-Pro-Ser-Pro-Ala-Val-Trp-Met-Ala- Arg-Asn-Thr 78) SEQ ID NO. 78: MAQSRLTATSASRVQ orMet-Ala-Gln-Ser-Arg-Leu-Thr-Ala-Thr-Ser-Ala-Ser- Arg-Val-Gln 79) SEQ ID NO. 79: AILLSQPPKQLGLRA orAla-lle-Leu-Leu-Ser-Gln-Pro-Pro-Lys-Gln-Leu-Gly- Leu-Arg-Ala 80) SEQ ID NO. 80: PANTPLIFVFSLEAG orPro-Ala-Asn-Thr-Pro-Leu-Ile-Phe-Val-Phe-Ser-Leu- Glu-Ala-Gly 81) SEQ ID NO. 81: FHHICQAGLKLLTSG orPhe-His-His-Ile-Cys-Gln-Ala-Gly-Leu-Lys-Leu-Leu- Thr-Ser-Gly 82) SEQ ID NO. 82: DPPASAFQSAGITGV orAsp-Pro-Pro-Ala-Ser-Ala-Phe-Gln-Ser-Ala-Gly-Ile- Thr-Gly-Val 83) SEQ ID NO. 83: SHLTQPANLDKKICS orSer-His-Leu-Thr-Gln-Pro-Ala-Asn-Leu-Asp-Lys-Lys- Ile-Cys-Ser 84) SEQ ID NO. 84: NGGSCYVAQAGLKLLASCNPSK orAsn-Gly-Gly-Ser-Cys-Tyr-Val-Ala-Gln-Ala-Gly-Leu-Lys-Leu-Leu-Ala-Ser-Cys-Asn-Pro-Ser-Lys  85) SEQ ID NO. 85:MWTLKSSLVLLLCLT or Met-Trp-Thr-Leu-Lys-Ser-Ser-Leu-Val-Leu-Leu-Leu-Cys-Leu-Thr  86) SEQ ID NO. 86: CSYAFMFSSLRQKTS orCys-Ser-Tyr-Ala-Phe-Met-Phe-Ser-Ser-Leu-Arg-Gln- Lys-Thr-Ser 87) SEQ ID NO. 87: EPQGKVPCGEHFRIR orGlu-Pro-Gln-Gly-Lys-Val-Pro-Cys-Gly-Glu-His-Phe- Arg-Ile-Arg 88) SEQ ID NO. 88: QNLPEHTQGWLGSKW orGln-Asn-Leu-Pro-Glu-His-Thr-Gln-Gly-Trp-Leu-Gly- Ser-Lys-Trp 89) SEQ ID NO. 89: LWLLFAVVPFVILKC orLeu-Trp-Leu-Leu-Phe-Ala-Val-Val-Pro-Phe-Val-Ile- Leu-Lys-Cys 90) SEQ ID NO. 90: QRDSEKNKVRMAPFF orGln-Arg-Asp-Ser-Glu-Lys-Asn-Lys-Val-Arg-Met-Ala- Pro-Phe-Phe 91) SEQ ID NO. 91: LHHIDSISGVSGKRMF orLeu-His-His-Ile-Asp-Ser-Ile-Ser-Gly-Val-Ser-Gly- Lys-Arg-Met-Phe 92) SEQ ID NO. 92: EAYYTMLHLPTTNRP orGlu-Ala-Tyr-Tyr-Thr-Met-Leu-His-Leu-Pro-Thr-Thr- Asn-Arg-Pro 93) SEQ ID NO. 93: KIAHCILFNQPHSPR orLys-Ile-Ala-His-Cys-Ile-Leu-Phe-Asn-Gln-Pro-His- Ser-Pro-Arg 94) SEQ ID NO. 94: SNSHSHPNPLKLHRR orSer-Asn-Ser-His-Ser-His-Pro-Asn-Pro-Leu-Lys-Leu- His-Arg-Arg 95) SEQ ID NO. 95: SHSHNRPRAYILITI orSer-His-Ser-His-Asn-Arg-Pro-Arg-Ala-Tyr-Ile-Leu- Ile-Thr-Ile 96) SEQ ID NO. 96: LPSKLKLRTHSQSHH orLeu-Pro-Ser-Lys-Leu-Lys-Leu-Arg-Thr-His-Ser-Gln- Ser-His-His 97) SEQ ID NO. 97: NPLSRTSNSTPTNSFLMTSSKPR orAsn-Pro-Leu-Ser-Arg-Thr-Ser-Asn-Ser-Thr-Pro-Thr-Asn-Ser-Phe-Leu-Met-Thr-Ser-Ser-Lys-Pro-Arg  98) SEQ ID NO. 98:SSSLGLPKCWDYRHE or Ser-Ser-Ser-Leu-Gly-Leu-Pro-Lys-Cys-Trp-Asp-Tyr-Arg-His-Glu  99) SEQ ID NO. 99: LLSLALMINFRVMAC orLeu-Leu-Ser-Leu-Ala-Leu-Met-Ile-Asn-Phe-Arg-Val- Met-Ala-Cys 100) SEQ ID NO. 100: TFKQHIELRQKISIV orThr-Phe-Lys-Gln-His-Ile-Glu-Leu-Arg-Gln-Lys-Ile- Ser-Ile-Val 101) SEQ ID NO. 101: PRKLCCMGPVCPVKI orPro-Arg-Lys-Leu-Cys-Cys-Met-Gly-Pro-Val-Cys-Pro- Val-Lys-Ile 102) SEQ ID NO. 102: ALLTINGHCTWLPAS orAla-Leu-Leu-Thr-Ile-Asn-Gly-His-Cys-Thr-Trp-Leu- Pro-Ala-Ser 103) SEQ ID NO. 103: MFVFCLILNREKIKG orMet-Phe-Val-Phe-Cys-Leu-Ile-Leu-Asn-Arg-Glu-Lys- Ile-Lys-Gly 104) SEQ ID NO. 104: GNSSFFLLSFFFSFQ orGly-Asn-Ser-Ser-Phe-Phe-Leu-Leu-Ser-Phe-Phe-Phe- Ser-Phe-Gln 105) SEQ ID NO. 105: NCCQCFQCRTTEGYA orAsn-Cys-Cys-Gln-Cys-Phe-Gln-Cys-Arg-Thr-Thr-Glu- Gly-Tyr-Ala 106) SEQ ID NO. 106: VECFYCLVDKAAFECWWFYSFDT orVal-Glu-Cys-Phe-Tyr-Cys-Leu-Val-Asp-Lys-Ala-Ala-Phe-Glu-Cys-Trp-Trp-Phe-Tyr-Ser-Phe-Asp-Thr  107) SEQ ID NO. 107:MEPHTVAQAGVPQHD or Met-Glu-Pro-His-Thr-Val-Ala-Gln-Ala-Gly-Val-Pro-Gln-His-Asp  108) SEQ ID NO. 108: LGSLQSLLPRFKRFS orLeu-Gly-Ser-Leu-Gln-Ser-Leu-Leu-Pro-Arg-Phe-Lys- Arg-Phe-Ser 109) SEQ ID NO. 109: CLILPKIWDYRNMNT orCys-Leu-Ile-Leu-Pro-Lys-Ile-Trp-Asp-Tyr-Arg-Asn- Met-Asn-Thr 110) SEQ ID NO. 110: ALIKRNRYTPETGRKS orAla-Leu-Ile-Lys-Arg-Asn-Arg-Tyr-Thr-Pro-Glu-Thr- Gly-Arg-Lys-Ser 111) SEQ ID NO. 111: IDQQVLSRI or Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile 112) SEQ ID NO. 112: KLEIKRCL or Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu 113) SEQ ID NO. 113: VLSRIK or Val-Leu-Ser-Arg-Ile-Lys 114) SEQ ID NO. 114: RIKLEIK or Arg-Ile-Lys-Leu-Glu-Ile-Lys 115) SEQ ID NO. 115: VLSRIKLEIKRCL orVal-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys- Leu,  and 116) SEQ ID NO. 116: IDQQVLSRIKLEI orIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu- Ile. The expression “NTP peptide” also preferably includes (but is notlimited to) the amino acid sequences of SEQ ID NO: 1 to 116.

The term “fragment” refers to a protein or polypeptide that consists ofa continuous subsequence of the amino acid sequence of a protein orpeptide and includes naturally occurring fragments such as splicevariants and fragments resulting from naturally occurring in vivoprotease activity. Such a fragment may be truncated at the aminoterminus, the carboxy terminus, and/or internally (such as by naturalsplicing). Such fragments may be prepared with or without an aminoterminal methionine. The term “fragment” includes fragments, whetheridentical or different, from the same protein or peptide, with acontiguous amino acid sequence in common or not, joined together, eitherdirectly or through a linker. A person having ordinary skill in the artwill be capable of selecting a suitable fragment for use in theembodiments without undue experimentation using the guidelines andprocedures outlined herein.

The term “variant” refers to a protein or polypeptide in which one ormore amino acid substitutions, deletions, and/or insertions are presentas compared to the amino acid sequence of an protein or peptide andincludes naturally occurring allelic variants or alternative splicevariants of an protein or peptide. The term “variant” includes thereplacement of one or more amino acids in a peptide sequence with asimilar or homologous amino acid(s) or a dissimilar amino acid(s). Thereare many scales on which amino acids can be ranked as similar orhomologous. (Gunnar von Heijne, Sequence Analysis in Molecular Biology,p. 123-39 (Academic Press, New York, N.Y. 1987.) Preferred variantsinclude alanine substitutions at one or more of amino acid positions.Other preferred substitutions include conservative substitutions thathave little or no effect on the overall net charge, polarity, orhydrophobicity of the protein. Conservative substitutions are set forthin Table 2 below.

TABLE 2 Conservative Amino Acid Substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Uncharged Polar: glutamineasparagine serine threonine tyrosine Non-Polar: phenylalanine tryptophancysteine glycine alanine valine praline methionine leucine isoleucine

Table 3 sets out another scheme of amino acid substitution:

TABLE 3 Original Residue Substitutions Ala gly;ser Arg lys Asn gln;hisAsp glu Cys ser Gln asn Glu asp Gly ala;pro His asn;gln Ile eu;val Leuile;val Lys arg;gln;glu Met leu;tyr;ile Phe met;leu;tyr Ser thr Thr serTrp tyr Tyr trp;phe Val ile;leu

Other variants can consist of less conservative amino acidsubstitutions, such as selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionsthat in general are expected to have a more significant effect onfunction are those in which (a) glycine and/or proline is substituted byanother amino acid or is deleted or inserted; (b) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) acysteine residue is substituted for (or by) any other residue; (d) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) a residue having an electronegativecharge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) one not havingsuch a side chain, e.g., glycine. Other variants include those designedto either generate a novel glycosylation and/or phosphorylation site(s),or those designed to delete an existing glycosylation and/orphosphorylation site(s). Variants include at least one amino acidsubstitution at a glycosylation site, a proteolytic cleavage site and/ora cysteine residue. Variants also include proteins and peptides withadditional amino acid residues before or after the protein or peptideamino acid sequence on linker peptides. For example, a cysteine residuemay be added at both the amino and carboxy terminals of an NTP peptidein order to allow the cyclisation of the peptide by the formation of adi-sulphide bond. The term “variant” also encompasses polypeptides thathave the amino acid sequence of an NTP peptide with at least one and upto 25 or more additional amino acids flanking either the 3′ or 5′ end ofthe peptide.

The term “derivative” refers to a chemically modified protein orpolypeptide that has been chemically modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques, as forexample, by addition of one or more polyethylene glycol molecules,sugars, phosphates, and/or other such molecules, where the molecule ormolecules are not naturally attached to wild-type proteins or NTPpeptides. Derivatives include salts. Such chemical modifications arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given protein or polypeptide. Also, a given protein orpolypeptide may contain many types of modifications. Modifications canoccur anywhere in a protein or polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, Proteins—Structure And Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,“Posttranslational Protein Modifications: Perspectives and Prospects,”pgs. 1-12 in Posttranslational Covalent Modification Of Proteins, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., “Protein Synthesis:Posttranslational Modifications and Aging,” Ann. N.Y. Acad. Sci. 663:48-62 (1992). The term “derivatives” include chemical modificationsresulting in the protein or polypeptide becoming branched or cyclic,with or without branching. Cyclic, branched and branched circularproteins or polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

The term “homologue” refers to a protein that is at least 60 percentidentical in its amino acid sequence of an NTP peptide as determined bystandard methods that are commonly used to compare the similarity inposition of the amino acids of two polypeptides. The degree ofsimilarity or identity between two proteins can be readily calculated byknown methods, including but not limited to those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo H.and Lipman, D., SIAM, J. Applied Math., 48:1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.

Preferred computer program methods useful in determining the identityand similarity between two sequences include, but are not limited to,the GCG program package (Devereux, J., et al., Nucleic Acids Research,12(1): 387 (1984)), BLASTP, BLASTN, and FASTA, Atschul, S. F. et al., J.Molec. Biol., 215: 403-410 (1990). The BLAST X program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol.Biol., 215: 403-410 (1990). By way of example, using a computeralgorithm such as GAP (Genetic Computer Group, University of Wisconsin,Madison, Wis.), the two proteins or polypeptides for which the percentsequence identity is to be determined are aligned for optimal matchingof their respective amino acids (the “matched span”, as determined bythe algorithm).

A gap opening penalty (which is calculated as 3 times the averagediagonal; the “average diagonal” is the average of the diagonal of thecomparison matrix being used; the “diagonal” is the score or numberassigned to each perfect amino acid match by the particular comparisonmatrix) and a gap extension penalty (which is usually 1/10 times the gapopening penalty), as well as a comparison matrix such as PAM 250 orBLOSUM 62 are used in conjunction with the algorithm. A standardcomparison matrix (see Dayhoff et al. in: Atlas of Protein Sequence andStructure, vol. 5, supp.3 for the PAM250 comparison matrix; see Henikoffet al., Proc. Natl. Acad. Sci USA, 89:10915-10919 for the BLOSUM 62comparison matrix) also may be used by the algorithm. The percentidentity then is calculated by the algorithm. Homologues will typicallyhave one or more amino acid substitutions, deletions, and/or insertionsas compared with the comparison protein or peptide, as the case may be.

The term “fusion protein” refers to a protein where one or more peptidesare recombinantly fused or chemically conjugated (including covalentlyand non-covalently) to a protein such as (but not limited to) anantibody or antibody fragment like an F.sub.ab fragment or short chainFv. The term “fusion protein” also refers to multimers (i.e. dimers,trimers, tetramers and higher multimers) of peptides. Such multimerscomprise homomeric multimers comprising one peptide, heteromericmultimers comprising more than one peptide, and heteromeric multimerscomprising at least one peptide and at least one other protein. Suchmultimers may be the result of hydrophobic, hyrdrophilic, ionic and/orcovalent associations, bonds or links, may be formed by cross-linksusing linker molecules or may be linked indirectly by, for example,liposome formation

The term “peptide mimetic” or “mimetic” refers to biologically activecompounds that mimic the biological activity of a peptide or a proteinbut are no longer peptidic in chemical nature, that is, they no longercontain any peptide bonds (that is, amide bonds between amino acids).Here, the term peptide mimetic is used in a broader sense to includemolecules that are no longer completely peptidic in nature, such aspseudo-peptides, semi-peptides and peptoids. Examples of peptidemimetics in this broader sense (where part of a peptide is replaced by astructure lacking peptide bonds) are described below. Whether completelyor partially non-peptide, peptide mimetics according to the embodimentsprovide a spatial arrangement of reactive chemical moieties that closelyresemble the three-dimensional arrangement of active groups in thepeptide on which the peptide mimetic is based. As a result of thissimilar active-site geometry, the peptide mimetic has effects onbiological systems that are similar to the biological activity of thepeptide.

The peptide mimetics of the embodiments are preferably substantiallysimilar in both three-dimensional shape and biological activity to thepeptides described herein. Examples of methods of structurally modifyinga peptide known in the art to create a peptide mimetic include theinversion of backbone chiral centers leading to D-amino acid residuestructures that may, particularly at the N-terminus, lead to enhancedstability for proteolytical degradation without adversely affectingactivity. An example is given in the paper “TritriatedD-ala.sup.1-Peptide T Binding”, Smith C. S. et al., Drug DevelopmentRes., 15, pp. 371-379 (1988). A second method is altering cyclicstructure for stability, such as N to C interchain imides and lactames(Ede et al. in Smith and Rivier (Eds.) “Peptides: Chemistry andBiology”, Escom, Leiden (1991), pp. 268-270). An example of this isgiven in conformationally restricted thymopentin-like compounds, such asthose disclosed in U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al.,the disclosure of which is incorporated by reference herein in itsentirety. A third method is to substitute peptide bonds in the peptideby pseudopeptide bonds that. confer resistance to proteolysis.

A number of pseudopeptide bonds have been described that in general donot affect peptide structure and biological activity. One example ofthis approach is to substitute retro-inverso pseudopeptide bonds(“Biologically active retroinverso analogues of thymopentin”, Sisto A.et al in Rivier, J. E. and Marshall, G. R. (eds) “Peptides, Chemistry,Structure and Biology”, Escom, Leiden (1990), pp. 722-773) and Dalpozzo,et al. (1993), Int. J. Peptide Protein Res., 41:561-566, incorporatedherein by reference). According to this modification, the amino acidsequences of the peptides may be identical to the sequences of anpeptide described above, except that one or more of the peptide bondsare replaced by a retro-inverso pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution willconfer resistance to proteolysis by exopeptidases acting on theN-terminus. Further modifications also can be made by replacing chemicalgroups of the amino acids with other chemical groups of similarstructure. Another suitable pseudopeptide bond that is known to enhancestability to enzymatic cleavage with no or little loss of biologicalactivity is the reduced isostere pseudopeptide bond (Couder, et al.(1993), Int. J. Peptide Protein Res., 41:181-184, incorporated herein byreference in its entirety).

Thus, the amino acid sequences of these peptides may be identical to thesequences of an peptide, except that one or more of the peptide bondsare replaced by an isostere pseudopeptide bond. Preferably the mostN-terminal peptide bond is substituted, since such a substitution wouldconfer resistance to proteolysis by exopeptidases acting on theN-terminus. The synthesis of peptides with one or more reduced isosterepseudopeptide bonds is known in the art (Couder, et al. (1993), citedabove). Other examples include the introduction of ketomethylene ormethylsulfide bonds to replace peptide bonds.

Peptoid derivatives of peptides represent another class of peptidemimetics that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.USA, 89:9367-9371, incorporated herein by reference in its entirety).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid (Simon, et al. (1992), cited above). Some or all ofthe amino acids of the peptides may be replaced with the N-substitutedglycine corresponding to the replaced amino acid.

The term “peptide mimetic” or “mimetic” also includes reverse-D peptidesand enantiomers as defined below.

The term “reverse-D peptide” refers to a biologically active protein orpeptide consisting of D-amino acids arranged in a reverse order ascompared to the L-amino acid sequence of an peptide. Thus, the carboxyterminal residue of an L-amino acid peptide becomes the amino terminalfor the D-amino acid peptide and so forth. For example, the peptide,ETESH (SEQ ID NO: 117 becomes H_(d)S_(d)E_(d)T_(d)E_(d), where E_(d),H_(d), S_(d), and T_(d) are the D-amino acids corresponding to theL-amino acids, E, H, S, and T respectively.

The term “enantiomer” refers to a biologically active protein or peptidewhere one or more the L-amino acid residues in the amino acid sequenceof an peptide is replaced with the corresponding D-amino acidresidue(s).

A “composition” as used herein, refers broadly to any compositioncontaining a recited peptide or amino acid sequence and, optionally anadditional active agent. The composition may comprise a dry formulation,an aqueous solution, or a sterile composition. Compositions comprisingpeptides may be employed as hybridization probes. The probes may bestored in freeze-dried form and may be associated with a stabilizingagent such as a carbohydrate. In hybridizations, the probe may bedeployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g., sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

The expression “clinically detected prostate cancer” denotes detectingprostate cancer clinically, and not through testing (e.g., PSA testing),or incidental detection. A person having ordinary skill in the art isfamiliar with the differences between clinical detection, incidentaldetection, and test-detection, and would know and understand thatclinically detected prostate cancer typically involves performing abiopsy of the affected or surrounding tissue and determining whetherthat tissue includes cancerous cells. See, e.g., S. Srivastava et al.,MOLECULAR PATHOLOGY OF EARLY CANCER, Chpt. 20 “Pathology and MolecularBiology of Early Prostate Cancer, Sakr and Grignon, pp. 301-320, IOSPress, 1999.

In an alternative embodiment in which an additional active agent is usedtogether with the NTP Peptide, the expression “active agent” is used todenote any agent capable of removing unwanted cellular proliferationsand/or tissue growth. Suitable active agents may include, but are notlimited to: (i) anti-cancer active agents (such as alkylating agents,topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNAantimetabolites, and antimitotic agents); (ii) active agents fortreating benign growths such as anti-acne and anti-wart active agents;(iii) antiandrogen compounds, (cyproterone acetate (1α,2β-methylene-6-chloro-17 α-acetoxy-6-dehydroprogesterone) Tamoxifen,aromatase inhibitors); (iv) alphal-adrenergic receptor blockers(tamsulosin, terazosin, doxazosin, prazosin, bunazosin, indoramin,alfulzosin, silodosin); (v) 5 α-reductase inhibitors (finasteride,dutasteride); (vi) phosphodiesterase type 5 (PDE5) inhibitors(tadalafil) and combinations thereof.

The embodiments are directed to methods of preventing or reducing theprogression of prostate cancer, and to methods of reducing the incidenceof prostate cancer in mammals comprising administering to a mammal inneed thereof a therapeutically effective amount of a compositioncomprising an NTP peptide, either alone or in combination with at leastone additional active agent capable of treating and/or killing unwantedcellular proliferations in mammals. In an embodiment, the mammalstreated are those that had not previously been diagnosed with clinicallydetected prostate cancer. In other embodiment, the mammals treated arethose that had been diagnosed with clinically detected prostate cancer.In another embodiment, the compositions are administered more than once.

Other peptide sequences derived from an NTP peptide found to be aneffective agent for causing cell death also may be used as an additionalactive agent in combination with the NTP peptides described herein. Aperson ordinarily skilled in the art can, using the guidelines providedherein, synthesize without undue experimentation fragments of aneffective Peptide spanning the entire amino acid sequence of thatprotein in order to identify other effective peptide sequences.

The inventor unexpectedly discovered that use of the NTP peptides intreating mammals in need of removal or destruction of unwanted cellularelements provided an unexpectedly superior reduction in the incidence ofclinically detected prostate cancer, and provided an unexpectedlyprevention or reduction in prostate cancer progression. The inventorunexpectedly discovered when conducting clinical trials for treating anunrelated disorder, BPH, that the administration of the NTP peptides,alone or in combination with another active agent, dramatically reducedthe incidence of clinically detected prostate cancer when compared tothe expected incidence in un-treated mammals. It has been reported thatlong term trial outcome results in mammals having BPH, have an incidenceof clinically detected prostate cancer of greater than about 20%.Thompson, I M, et al., “The Influence of Finasteride on the Developmentof Prostate Cancer,” N Engl J Med, Vol. 349, pp. 215-224 (2003);Andriole G L, et al., “Effect of Dutasteride on the Risk of ProstateCancer,” N Engl J Med, Vol. 362, pp. 1192-1202 (2010). The inventordiscovered during clinical trials that the incidence of clinicallydetected prostate cancer in men suffering from BPH, when treated with acontrol (vehicle alone) was from about 5-6%.

In contrast to the published literature and the controls from theclinical trial, patients treated with the compositions of the presentinvention exhibited a dramatic decrease in the incidence of clinicallydetected prostate cancer. The method of the embodiments can prevent theincidence of clinically detected prostate cancer (incidence is 0%), orreduce the incidence rate, when compared to controls, by more than 15%,or by more than 20%, or by more than 25%, or by more than 30%, or bymore than 40%, or by more than 50%, and by orders of magnitude, whencompared to the known literature. The embodiments may result in anincidence of developing clinically detected prostate cancer within therange of from about 0% to about 5%, or from about 0% to about 4%, orfrom about 0% to about 3%, or from about 0% to about 2%, or from about0% to about 1.5%, or from about 0% to about 1.3%, or from about 0% toabout 1.0%, or from about 0% to about 0.8%.

The inventor also unexpectedly discovered when conducting clinicaltrials for treating an unrelated disorder, BPH, that the administrationof the NTP peptides, alone or in combination with another active agent,dramatically reduced and/or prevented the progression of prostate cancerin patients at risk of developing prostate cancer. A patient at risk ofdeveloping prostate cancer is a patient having test-detected prostatecancer, such as having an elevated PSA level. In one embodiment,patients at risk of developing prostate cancer have a baseline PSA ofmore than 2.0, or more than 2.3, or more than 2.5. The inventordiscovered that, for this subpopulation of patients, that administeringthe compositions of the embodiments resulted in a reduction in theincidence of clinically detected prostate cancer of from about 0% toless than about 5%, or from about 0% to less than about 4%, or fromabout 0% to less than about 3%, or from about 0% to less than about 2%,or from about 0% to less than about 1%, or from about 0% to about 0.5%,or from about 0% to about 0.2%, or about 0% (denoting prevention ofprogression of prostate cancer).

The embodiments include a method of treating a mammal suffering from acondition requiring the removal or destruction of unwanted cellularproliferations, comprising administering once or more than once an NTPpeptide to the mammal, either alone or in combination withadministration of an additional active agent. The method includes, butis not limited to, administering the NTP-peptides intramuscularly,orally, intravenously, intraperitoneally, intracerebrally(intraparenchymally), intracerebroyentricularly, intralesionally,intraocularly, intraarterially, intrathecally, intratumorally,intranasally, topically, transdermally, subcutaneously, orintradermally, either alone or conjugated to a carrier. The unwantedcellular proliferations include, inter alia, benign and malignanttumors, glandular (e.g. prostate) hyperplasia, and cancer. Preferred NTPpeptides include one or more of the following:

SEQ ID No. 66 IDQQVLSRIKLEIKRCLIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu- Ile-Lys-Arg-Cys-Leu SEQ ID NO. 111 IDQQVLSRI  Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile SEQ ID NO. 115 VLSRIKLEIKRCLVal-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-  Leu SEQ ID NO. 116IDQQVLSRIKLEI Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-  Ile

Any mammal can benefit from use of the invention, including humans,mice, rabbits, dogs, sheep and other livestock, any mammal treated ortreatable by a veterinarian, zoo-keeper, or wildlife preserve employee.Preferred mammals are humans, sheep, and dogs. Throughout thisdescription mammals and patients are used interchangeably.

It will be apparent to one of skill in the art that other smallerfragments of the above NTP peptides may be selected such that thesepeptides will possess the same or similar biological activity. Otherfragments of may be selected by one skilled in the art such that thesepeptides will possess the same or similar biological activity. Thepeptides of the embodiments encompass these other fragments. In general,the peptides of the embodiments have at least 4 amino acids, preferablyat least 5 amino acids, and more preferably at least 6 amino acids.

The embodiments also encompass methods of treating mammals (or patients)in need of removal or destruction of unwanted cellular proliferationscomprising administering a composition comprising NTP peptidescomprising two or more NTP peptides joined together, together with anadditional active agent. To the extent that an NTP peptide has thedesired biological activity, it follows that two such Peptides wouldalso possess the desired biological activity.

NTP peptides and fragments, variants, derivatives, homologues, fusionproteins and mimetics thereof encompassed by this embodiment can beprepared using methods known to those of skill in the art, such asrecombinant DNA technology, protein synthesis and isolation of naturallyoccurring peptides, proteins, AD7c-protein and fragments, variants,derivatives and homologues thereof.

NTP peptides and fragments, variants, derivatives, homologues, fusionproteins and mimetics thereof can be prepared from other peptides,proteins, and fragments, variants, derivatives and homologues thereofusing methods known to those having skill in the art. Such methodsinclude (but are not limited to) the use of proteases to cleave thepeptide, or protein into the desired NTP peptides.

An NTP peptide can be prepared using well known recombinant DNAtechnology methods such as those set forth in Sambrook et al. MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. and/or Ausubel et al., eds., Current Protocols inMolecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y.

A gene or cDNA encoding an NTP peptide may be obtained for example byscreening a genomic or cDNA library, or by PCR amplification. Probes orprimers useful for screening the library can be generated based onsequence information for other known genes or gene fragments from thesame or a related family of genes, such as, for example, conservedmotifs found in other peptides or proteins. In addition, where a geneencoding an NTP peptide has been identified, all or a portion of thatgene may be used as a probe to identify homologous genes. The probes orprimers may be used to screen cDNA libraries from various tissue sourcesbelieved to express an NTP peptide gene. Typically, conditions of highstringency will be employed for screening to minimize the number offalse positives obtained from the screen.

Another means to prepare a gene encoding an NTP peptide is to employchemical synthesis using methods well known to the skilled artisan, suchas those described by Engels et al., Angew. Chem. Intl. Ed., 28:716-734.These methods include, inter alia, the phosphotriester, phosphoramidite,and H-phosphonate methods for nucleic acid synthesis. A preferred methodfor such chemical synthesis is polymer-supported synthesis usingstandard phosphoramidite chemistry. Typically, the DNA encoding anpeptide or protein will be several hundred nucleotides in length.Nucleic acids larger than about 100 to nucleotides can be synthesized asseveral fragments using these methods. The fragments then can be ligatedtogether to form the full length peptide or protein. Usually, the DNAfragment encoding the amino terminus of the protein will have an ATG,which encodes a methionine residue. This methionine may or may not bepresent on the mature form of the protein or peptide, depending onwhether the protein produced in the host cell is designed to be secretedfrom that cell.

The gene, cDNA, or fragment thereof encoding the NTP peptide can beinserted into an appropriate expression or amplification vector usingstandard ligation techniques. The vector is typically selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene can occur). The gene, cDNA orfragment thereof encoding the NTP peptide may be amplified/expressed inprokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic hostcells. Selection of the host cell will depend in part on whether the NTPpeptide is to be glycosylated and/or phosphorylated. If so, yeast,insect, or mammalian host cells are preferable.

Typically, the vectors used in any of the host cells will contain atleast a 5′ flanking sequence (also referred to as a promoter) and otherregulatory elements as well, such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a completeintron sequence containing a donor and acceptor splice site, a signalpeptide sequence, a ribosome binding site element, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these elements is discussed below. Optionally, the vector may containa tag sequence, i.e., an oligonucleotide molecule located at the 5′ or3′ end of the protein or peptide coding sequence; the oligonucleotidemolecule encodes polyHis (such as hexaHis (SEQ ID NO: 118)), or othertag such as FLAG, HA (hemaglutinin Influenza virus) or myc for whichcommercially available antibodies exist. This tag is typically fused tothe polypeptide upon expression of the polypeptide, and can serve asmeans for affinity purification of the protein or peptide from the hostcell. Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purifiedprotein or peptide by various means such as using certain peptidases.

The human immunoglobulin hinge and Fc region could be fused at eitherthe N-terminus or C-terminus of the NTP peptide by one skilled in theart. The subsequent Fc-fusion protein could be purified by use of aProtein A affinity column. Fc is known to exhibit a long pharmacokinetichalf-life in vivo and proteins fused to Fc have been found to exhibit asubstantially greater half-life in vivo than the unfused counterpart.Also, fusion to the Fc region allows for dimerization/multimerization ofthe molecule that may be useful for the bioactivity of some molecules.

The 5′ flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5′ flanking sequences from more than one source), synthetic, or itmay be the native protein or peptide gene 5′ flanking sequence. As such,the source of the 5′ flanking sequence may be any unicellularprokaryotic or eukaryotic organism, any vertebrate or invertebrateorganism, or any plant, provided that the 5′ flanking sequence isfunctional in, and can be activated by, the host cell machinery.

The 5′ flanking sequences useful in the vectors of this embodiment maybe obtained by any of several methods well known in the art. Typically,5′ flanking sequences useful herein other than the protein or peptidegene flanking sequence will have been previously identified by mappingand/or by restriction endonuclease digestion and can thus be isolatedfrom the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of the 5′flanking sequence may be known. Here, the 5′ flanking sequence may besynthesized using the methods described above for nucleic acid synthesisor cloning. Where all or only a portion of the 5′ flanking sequence isknown, it may be obtained using PCR and/or by screening a genomiclibrary with suitable oligonucleotide and/or 5′ flanking sequencefragments from the same or another species.

Where the 5′ flanking sequence is not known, a fragment of DNAcontaining a 5′ flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, Qiagen® column or other methodsknown to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe protein or peptide. If the vector of choice does not contain anorigin of replication site, one may be chemically synthesized based on aknown sequence, and ligated into the vector. The transcriptiontermination element is typically located 3′ of the end of the protein orpeptide coding sequence and serves to terminate transcription of theprotein or peptide. Usually, the transcription termination element inprokaryotic cells is a G-C rich fragment followed by a poly T sequence.While the element may be cloned from a library or purchased commerciallyas part of a vector, it can also be readily synthesized using methodsfor nucleic acid synthesis such as those described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translation initiation of mRNA. The element is typicallylocated 3′ to the promoter and 5′ to the coding sequence of the proteinor peptide to be synthesized. The Shine-Dalgarno sequence is varied butis typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for an NTP peptide to be secretedfrom the host cell, a signal sequence may be used to direct the Peptideout of the host cell where it is synthesized, and the carboxy-terminalpart of the protein may be deleted in order to prevent membraneanchoring. Typically, the signal sequence is positioned in the codingregion of the NTP peptide gene or cDNA, or directly at the 5′ end of thePeptide gene coding region. Many signal sequences have been identified,and any of them that are functional in the selected host cell may beused in conjunction with the Peptide gene or cDNA. Therefore, the signalsequence may be homologous or heterologous to the Peptide gene or cDNA,and may be homologous or heterologous to the Peptide gene or cDNA.Additionally, the signal sequence may be chemically synthesized usingmethods set forth above. In most cases, secretion of the polypeptidefrom the host cell via the presence of a signal peptide will result inthe removal of the amino terminal methionine from the polypeptide.

In many cases, transcription of the NTP peptide gene or cDNA isincreased by the presence of one or more introns in the vector; this isparticularly true where the Peptide is produced in eukaryotic hostcells, especially mammalian host cells. The introns used may benaturally occurring within the Peptide gene, especially where the geneused is a full length genomic sequence or a fragment thereof. Where theintron is not naturally occurring within the gene (as for most cDNAs),the intron(s) may be obtained from another source. The position of theintron with respect to the flanking sequence and the Peptide genegenerally is important, as the intron must be transcribed to beeffective. As such, where the Peptide gene inserted into the expressionvector is a cDNA molecule, the preferred position for the intron is 3′to the transcription start site, and 5′ to the polyA transcriptiontermination sequence. Preferably for Peptide cDNA, the intron or intronswill be located on one side or the other (i.e., 5′ or 3′) of the cDNAsuch that it does not interrupt this coding sequence. Any intron fromany source, including any viral, prokaryotic and eukaryotic (plant oranimal) organisms, may be used to practice this embodiment, providedthat it is compatible with the host cell(s) into which it is inserted.Also included herein are synthetic introns. Optionally, more than oneintron may be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this embodiment may be constructedfrom starting vectors such as a commercially available vector. Suchvectors may or may not contain some of the elements to be included inthe completed vector. If none of the desired elements are present in thestarting vector, each element may be individually ligated into thevector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to blunt the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in “sticky ends” using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra. Alternatively, two or more of the elements to be inserted intothe vector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

An additional method for constructing the vector is to conduct allligations of the various elements simultaneously in one reactionmixture. Here, many nonsense or nonfunctional vectors will be generateddue to improper ligation or insertion of the elements, however thefunctional vector may be identified and selected by restrictionendonuclease digestion.

Preferred vectors for practicing this embodiment are those that arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15b (Novagen, Madison, Wis.), PGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBachl; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding full length or truncated protein or peptide has been insertedinto the proper site of the vector, the completed vector may be insertedinto a suitable host cell for amplification and/or polypeptideexpression. Host cells may be prokaryotic host cells (such as E. coli)or eukaryotic host cells (such as a yeast cell, an insect cell, or avertebrate cell). The host cell, when cultured under appropriateconditions, can synthesize protein or peptide which can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted).

After collection, the NTP peptide can be purified using methods such asmolecular sieve chromatography, affinity chromatography, and the like.Selection of the host cell for protein or peptide production will dependin part on whether the Peptide is to be glycosylated or phosphorylated(in which case eukaryotic host cells are preferred), and the manner inwhich the host cell is able to fold the Peptide into its native tertiarystructure (e.g., proper orientation of disulfide bridges, etc.) suchthat biologically active protein is prepared by the Peptide that hasbiological activity, the Peptide may be folded after synthesis usingappropriate chemical conditions as discussed below. Suitable cells orcell lines may be mammalian cells, such as Chinese hamster ovary cells(CHO), human embryonic kidney (HEK) 293, 293T cells, or 3T3 cells. Theselection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. Other suitable mammalian celllines, are the monkey COS-1 and COS-7 cell lines, and the CV-1 cellline. Further exemplary mammalian host cells include primate cell linesand rodent cell lines, including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain adominantly acting selection gene. Other suitable mammalian cell linesinclude but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHKor HaK hamster cell lines.

Similarly useful as host cells suitable for the present embodiments arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5.alpha., DH10, and MC1061) are well-known as host cells in thefield of biotechnology. Various strains of B. subtilis, Pseudomonasspp., other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method. Many strains of yeast cells known to thoseskilled in the art also are available as host cells for expression ofthe polypeptides of the present embodiments.

Additionally, where desired, insect cell systems may be utilized in themethods of the present embodiments. Such systems are described forexample in Kitts et al. (Biotechniques, 14:810-817), Lucklow (Curr.Opin. Biotechnol., 4:564-572) and Lucklow et al. (J. Virol.,67:4566-4579). Preferred insect cells are Sf-9 and Hi5 (Invitrogen,Carlsbad, Calif.).

Insertion (also referred to as transformation or transfection) of thevector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection, or the DEAE-dextran method. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The media will usually contain all nutrients necessary for the growthand survival of the cells. Suitable media for culturing E. coli cellsare for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitablemedia for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all ofwhich may be supplemented with serum and/or growth factors as requiredby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary. Typically, anantibiotic or other compound useful for selective growth of thetransformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of NTP peptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,mass spectroscopy, immunoprecipitation, and/or activity assays such asDNA binding gel shift assays.

If the protein or peptide has been designed to be secreted from the hostcells, the majority of the protein or peptide may be found in the cellculture medium. Proteins prepared in this way will typically not possessan amino terminal methionine, as it is removed during secretion from thecell. If however, the protein or peptide is not secreted from the hostcells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the cytosol (for gram negative bacteriahost cells) and may have an amino terminal methionine.

For an NTP peptide situated in the host cell cytoplasm and/or nucleus,the host cells are typically first disrupted mechanically or withdetergent to release the intra-cellular contents into a bufferedsolution. The Peptide can then be isolated from this solution.

Purification of NTP peptides from solution can be accomplished using avariety of techniques. If the NTP peptide has been synthesized such thatit contains a tag such as hexaHistidine (SEQ ID NO: 118) (e.g.peptide/hexaHis (SEQ ID NO: 118)) or other small peptide such as FLAG(Sigma-Aldritch, St. Louis, Mo.) or calmodulin-binding peptide(Stratagene, La Jolla, Calif.) at either its carboxyl or amino terminus,it may essentially be purified in a one-step process by passing thesolution through an affinity column where the column matrix has a highaffinity for the tag or for the protein directly (i.e., a monoclonalantibody specifically recognizing the peptide). For example,polyhistidine binds with great affinity and specificity to nickel, zincand cobalt; thus immobilized metal ion affinity chromatography whichemploys a nickel-based affinity resin (as used in Qiagen's QlAexpresssystem or Invitrogen's Xpress System) or a cobalt-based affinity resin(as used in BD Biosciences-CLONTECH's Talon system) can be used forpurification of peptide/polyHis. (See, for example, Ausubel et al.,eds., Current Protocols in Molecular Biology, Section 10.11.8, JohnWiley & Sons, New York).

Where the NTP peptide is prepared without a tag attached, and noantibodies are available, other well known procedures for purificationcan be used. Such procedures include, without limitation, ion exchangechromatography, hydroxyapatite chromatography, hydrophobic interactionchromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (Isoprime machine/technique, Hoefer Scientific). Insome cases, two or more of these techniques may be combined to achieveincreased purity.

If it is anticipated that the NTP peptide will be found primarilyintracellularly, the intracellular material (including inclusion bodiesfor gram-negative bacteria) can be extracted from the host cell usingany standard technique known to the skilled artisan. For example, thehost cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation. If the Peptide has formed inclusion bodiesin the cytosol, the inclusion bodies can often bind to the inner and/orouter cellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material then can be treatedat pH extremes or with chaotropic agent such as a detergent, guanidine,guanidine derivatives, urea, or urea derivatives in the presence of areducing agent such as dithiothreitol at alkaline pH or triscarboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The Peptide in its now soluble form canthen be analyzed using gel electrophoresis, immunoprecipitation or thelike. If it is desired to isolate the Peptide, isolation may beaccomplished using standard methods such as those set forth below and inMarston et al. Meth. Enz., 182:264-275.

In some cases, the NTP peptide may not be biologically active uponisolation. Various methods for refolding or converting the polypeptideto its tertiary structure and generating disulfide linkages, can be usedto restore biological activity. Such methods include exposing thesolubilized polypeptide to a pH usually above 7 and in the presence of aparticular concentration of a chaotrope. The selection of chaotrope isvery similar to the choices used for inclusion body solubilization butusually at a lower concentration and is not necessarily the samechaotrope as used for the solubilization. In most cases therefolding/oxidation solution will also contain a reducing agent or thereducing agent plus its, oxidized form in a specific ratio to generate aparticular redox potential allowing for disulfide shuffling to occur inthe formation of the protein's cysteine bridge(s). Some of the commonlyused redox couples include cysteine/cystamine, glutathione(GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT,2-mercaptoethanol(bME)/dithio-b(ME). In many instances a cosolvent isnecessary to increase the efficiency of the refolding and the morecommon reagents used for this purpose include glycerol, polyethyleneglycol of various molecular weights, and arginine.

If NTP peptide inclusion bodies are not formed to a significant degreein the host cell, the NTP peptide will be found primarily in thesupernatant after centrifugation of the cell homogenate, and the NTPpeptide can be isolated from the supernatant using methods such as thoseset forth below.

In those situations where it is preferable to partially or completelyisolate the NTP peptide, purification can be accomplished using standardmethods well known to the skilled artisan. Such methods include, withoutlimitation, separation by electrophoresis followed by electroelution,various types of chromatography (immunoaffinity, molecular sieve, and/orion exchange), and/or high pressure liquid chromatography. In somecases, it may be preferable to use more than one of these methods forcomplete purification.

In addition to preparing and purifying NTP peptides using recombinantDNA techniques, the NTP peptides and their fragments, variants,homologues, fusion proteins, peptide mimetics, and derivatives may beprepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art such as those set forth byMerrifield et al., J. Am. Chem. Soc., 85:2149, Houghten et al. Proc NatlAcad. Sci. USA, 82:5132, and Stewart and Young, Solid Phase PeptideSynthesis, Pierce Chemical Co., Rockford, Ill. Such Peptides may besynthesized with or without a methionine on the amino terminus.Chemically synthesized NTP peptides may be oxidized using methods setforth in these references to form disulfide bridges. The NTP peptidesare expected to have biological activity comparable to Peptides producedrecombinantly or purified from natural sources, and thus may be usedinterchangeably with recombinant or natural Peptide.

Chemically modified NTP peptide compositions in which the Peptide islinked to a polymer are included within the scope of the presentembodiments. The polymer selected is typically water soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. The polymer selectedis usually modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled as provided for in the present methods.The polymer may be of any molecular weight, and may be branched orunbranched. Included within the scope of peptide polymers is a mixtureof polymers.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of the naturally occurring NTP peptides. Nucleic acidvariants may be produced using site directed mutagenesis, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques). Chemicalsynthesis using methods described by Engels et al., supra, may also beused to prepare such variants. Other methods known to the skilledartisan may be used as well.

Preferred nucleic acid variants are those containing nucleotidesubstitutions accounting for codon preference in the host cell that isto be used to produce NTP peptides. Such codon optimization can bedetermined via computer algorithers which incorporate codon frequencytables such as Ecohigh. Cod for codon preference of highly expressedbacterial genes as provided by the University of Wisconsin PackageVersion 9.0, Genetics Computer Group, Madison, Wis. Other useful codonfrequency tables include Celegans_high.cod, Celegans_low.cod,Drosophila_high.cod, Human_high.cod, Maize_high.cod, and Yeast_high.cod.Other preferred variants are those encoding conservative amino acidchanges as described above (e.g., wherein the charge or polarity of thenaturally occurring amino acid side chain is not altered substantiallyby substitution with a different amino acid) as compared to wild type,and/or those designed to either generate a novel glycosylation and/orphosphorylation site(s), or those designed to delete an existingglycosylation and/or phosphorylation site(s).

NTP peptides and fragments, homologs, variants, fusion proteins, peptidemimetics, derivatives and salts thereof also can be made usingconventional peptide synthesis techniques known to the skilled artisan.These techniques include chemical coupling methods (cf. Wunsch, E:“Methoden der organischen Chemie”, Volume 15, Band 1+2, Synthese vonPeptiden, thime Verlag, Stuttgart (1974), and Barrany, G.; Merrifield,R. B.: “The Peptides,” eds. E. Gross, J. Meienhofer, Volume 2, Chapter1, pp. 1-284, Academic Press (1980)), enzymatic coupling methods (cf.Widmer, F. Johansen, J. T., Carlsberg Res. Commun., Vol. 44, pp. 37-46(1979); Kullmann, W.: “Enzymatic Peptide Synthesis”, CRC Press Inc. BocaRaton, Fla. (1987); and Widmer, F., Johansen, J. T. in “SyntheticPeptides in Biology and Medicines,” eds. Alitalo, K., Partanen, P.,Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985)), or a combination ofchemical and enzymatic methods if this is advantageous for the processdesign and economy. Using the guidelines provided herein, those skilledin the art are capable of varying the peptide sequence of the NTPpeptide to make a homologue having the same or similar biologicalactivity (bioactivity) as the original or native NTP peptide.

Advantages exist for using a mimetic of a given NTP peptide rather thanthe Peptide itself. In general, peptide mimetics are more bioavailable,have a longer duration of action and can be cheaper to produce than thenative proteins and peptides.

Peptide mimetics of NTP peptides can be developed using combinatorialchemistry techniques and other techniques known in the art (see e.g.Proceedings of the 20th European Peptide Symposium, ed. G. Jung, E.Bayer, pp. 289-336, and references therein). Examples of methods knownin the art for structurally modifying a peptide to create a peptidemimetic include the inversion of backbone chiral centers leading toD-amino acid residue structures that may, particularly at theN-terminus, lead to enhanced stability for proteolytical degradationwithout adversely affecting activity. An example is provided in thepaper “Tritriated D-ala.sup.1-Peptide T Binding”, Smith C. S. et al.,Drug Development Res. 15, pp. 371-379 (1988).

A second method is altering cyclic structure for stability, such as N toC interchain imides and lactames (Ede et al. in Smith and Rivier (Eds.)“Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270).An example of this is given in conformationally restrictedthymopentin-like compounds, such as those disclosed in U.S. Pat. No.4,457,489 (1985), Goldstein, G. et al., the disclosure of which isincorporated by reference herein in its entirety.

A third method is to substitute peptide bonds in the NTP peptide bypseudopeptide bonds that confer resistance to proteolysis. A number ofpseudopeptide bonds have been described that in general do not affectpeptide structure and biological activity. One example of this approachis to substitute retro-inverso pseudopeptide bonds (“Biologically activeretroinverso analogues of thymopentin”, Sisto A. et al in Rivier, J. E.and Marshall, G. R. (eds) “Peptides, Chemistry, Structure and Biology”,Escom, Leiden (1990), pp. 722-773) and Dalpozzo, et al. (1993), Int. J.Peptide Protein Res., 41:561-566, incorporated herein by reference).According to this modification, the amino acid sequences of the peptidesmay be identical to the sequences of the peptides described above,except that one or more of the peptide bonds are replaced by aretro-inverso pseudopeptide bond. Preferably the most N-terminal peptidebond is substituted, since such a substitution will confer resistance toproteolysis by exopeptidases acting on the N-terminus.

The synthesis of peptides with one or more reduced retro-inversopseudopeptide bonds is known in the art (Sisto (1990) and Dalpozzo, etal. (1993), cited above). Thus, peptide bonds can be replaced bynon-peptide bonds that allow the peptide mimetic to adopt a similarstructure, and therefore biological activity, to the original peptide.Further modifications also can be made by replacing chemical groups ofthe amino acids with other chemical groups of similar structure. Anothersuitable pseudopeptide bond that is known to enhance stability toenzymatic cleavage with no or little loss of biological activity is thereduced isostere pseudopeptide bond is a (Couder, et al. (1993), Int. J.Peptide Protein Res., 41:181-184, incorporated herein by reference inits entirety). Thus, the amino acid sequences of these peptides may beidentical to the sequences of an peptide, except that one or more of thepeptide bonds are replaced by an isostere pseudopeptide bond. Preferablythe most N-terminal peptide bond is substituted, since such asubstitution would confer resistance to proteolysis by exopeptidasesacting on the N-terminus. The synthesis of peptides with one or morereduced isostere pseudopeptide bonds is known in the art (Couder, et al.(1993), cited above). Other examples include the introduction ofketomethylene or methylsulfide bonds to replace peptide bonds.

Peptoid derivatives of NTP peptides represent another class of peptidemimetics that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.USA, 89:9367-9371 and incorporated herein by reference in its entirety).Peptoids are oligomers of N-substituted glycines. A number of N-alkylgroups have been described, each corresponding to the side chain of anatural amino acid (Simon, et al. (1992), cited above and incorporatedherein by reference in its entirety). Some or all of the amino acids ofthe peptide are replaced with the N-substituted glycine corresponding tothe replaced amino acid.

The development of peptide mimetics can be aided by determining thetertiary structure of the original peptide by NMR spectroscopy,crystallography and/or computer-aided molecular modeling. Thesetechniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal peptide (Dean (1994), BioEssays, 16: 683-687; Cohen andShatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993),Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15:124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993),Sci. Am., 269: 92-98, all incorporated herein by reference in theirentirety).

Once a potential peptide mimetic compound is identified, it may besynthesized and assayed using the methods outlined in the examples belowto assess its activity. The peptide mimetic compounds obtained by theabove methods, having the biological activity of the peptides andsimilar three-dimensional structure, are encompassed by this embodiment.It will be readily apparent to one skilled in the art that a peptidemimetic can be generated from any of the peptides bearing one or more ofthe modifications described above. It will furthermore be apparent thatthe peptide mimetics of this embodiment can be further used for thedevelopment of even more potent non-peptidic compounds, in addition totheir utility as therapeutic compounds.

A number of organizations exist today that are capable of synthesizingthe peptides described herein. For example, given the sequence of an NTPpeptide, the organization can synthesize the Peptide and forward thesynthesized Peptide with accompanying documentation and proof of theidentity of the Peptide.

The present embodiments are directed to methods of treating mammals withconditions requiring removal of cells, such as benign and malignanttumors, glandular (e.g. prostate) hyperplasia, cancer in which thetreatment reduces the incidence of clinically developed prostate cancer,or prevents or reduces the progression of prostate cancer in patients atrisk of developing prostate cancer. Such a method comprisesadministering to a mammal in need thereof, a therapeutically effectiveamount of NTP peptide, either alone, or in combination with anadditional active agent. The mammals in need may be mammals sufferingfrom benign prostatic hyperplasia, that may or may not be at increasedrisk of developing prostate cancer, or mammals at risk of developingprostate cancer. The mammals in need also may be any mammal that wouldbenefit from a reduction in the incidence of clinically detectedprostate cancer.

The additional active agent, if used, can be one or more active agentsselected from (i) anti-cancer active agents (such as alkylating agents,topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNAantimetabolites, and antimitotic agents); (ii) active agents fortreating benign growths such as anti-acne and anti-wart active agents(salicylic acid); (iii) antiandrogen compounds, (cyproterone acetate(1α, 2β-methylene-6-chloro-17 α-acetoxy-6-dehydroprogesterone))Tamoxifen, aromatase inhibitors); (iv) alphal-adrenergic receptorblockers (tamsulosin, terazosin, doxazosin, prazosin, bunazosin,indoramin, alfulzosin, silodosin); (v) 5 α-reductase inhibitors(finasteride, dutasteride); (vi) phosphodiesterase type 5 (PDE5)inhibitors (tadalafil) and combinations thereof. Preferably, theadditional active agent is selected from the group consisting oftamsulosin, finasteride, terazosin, doxazosin, prazosin, tadalafil,alfuzosin, silodosin, dutasteride, combinations of dutasteride andtamsulosin, and mixtures and combinations thereof.

The condition can be, for example, tumors of lung, breast, stomach,pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon,intestine, stomach, rectum, esophagus, blood, brain and its coverings,spinal cord and its coverings, muscle, connective tissue, adrenal,parathyroid, thyroid, uterus, testis, pituitary, reproductive organs,liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, lymph nodesand lymphoid system, and other organs. In certain embodiments, thecondition is benign prostatic hyperplasia, and in other embodiments, thecondition is prostate cancer.

As used herein, the term “malignant tumor” is intended to encompass allforms of human carcinomas, sarcomas and melanomas which occur in thepoorly differentiated, moderately differentiated, andwell-differentiated forms.

The condition to be treated can also be a hyperplasia, hypertrophy, orovergrowth of a tissue selected from the group consisting of lung,breast, stomach, pancreas, prostate, bladder, bone, ovary, skin, kidney,sinus, colon, intestine, stomach, rectum, esophagus, blood, brain andits coverings, spinal cord and its coverings, muscle, connective tissue,adrenal, parathyroid, thyroid, uterus, testis, pituitary, reproductiveorgans, liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, andlymph nodes and lymphoid system.

Other conditions that can be treated using the method of the embodimentsare virally, bacterially, or parasitically altered tissue selected fromthe group consisting of lung, breast, stomach, pancreas, prostate,bladder, bone, ovary, skin, kidney, sinus, colon, intestine, stomach,rectum, esophagus, blood, brain and its coverings, spinal cord and itscoverings, muscle, connective tissue, adrenal, parathyroid, thyroid,uterus, testis, pituitary, reproductive organs, liver, gall bladder,eye, ear, nose, throat, tonsils, mouth, and lymph nodes and lymphoidsystem.

The condition to be treated can also be a malformation or disorder of atissue selected from the group consisting of lung, breast, stomach,pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon,intestine, stomach, rectum, esophagus, blood, brain and its coverings,spinal cord and its coverings, muscle, connective tissue, adrenal,parathyroid, thyroid, uterus, testis, pituitary, reproductive organs,liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, and lymphnodes and lymphoid system.

In particular, the condition to be treated can be tonsillar hypertrophy,prostatic hyperplasia, psoriasis, eczema, dermatoses or hemorrhoids. Thecondition to be treated can be a vascular disease, such asatherosclerosis or arteriosclerosis, or a vascular disorder, such asvaricose veins, stenosis or restenosis of an artery or a stent. Thecondition to be treated can also be a cosmetic modification to a tissue,such as skin, eye, ear, nose, throat, mouth, muscle, connective tissue,hair, or breast tissue.

Therapeutic compositions of NTP peptides may comprise a therapeuticallyeffective amount of an NTP peptide in admixture with a pharmaceuticallyacceptable carrier. In some alternative embodiments, the additionalactive agent can be administered in the same composition with the NTPpeptide, and in other embodiments, the composition comprising the NTPpeptide is administered as an injection, whereas the additional activeagent is formulated into an oral medication (gel, capsule, tablet,liquid, etc.). The carrier material may be water for injection,preferably supplemented with other materials common in solutions foradministration to mammals. Typically, an NTP peptide for therapeutic usewill be administered in the form of a composition comprising purifiedpeptide in conjunction with one or more physiologically acceptablecarriers, excipients, or diluents. Neutral buffered saline or salinemixed with serum albumin are exemplary appropriate carriers. Preferably,the product is formulated as a lyophilizate using appropriate excipients(e.g., sucrose). Other standard carriers, diluents, and excipients maybe included as desired. Compositions of the embodiments also maycomprise buffers known to those having ordinary skill in the art with anappropriate range of pH values, including Tris buffer of about pH7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may furtherinclude sorbitol or a suitable substitute therefor.

The use of NTP peptides conjugated or linked or bound to an antibody,antibody fragment, antibody-like molecule, or a molecule with a highaffinity to a specific tumor marker, such as a cellular receptor, signalpeptide or over-expressed enzyme, for targeting to the unwanted cellularelements in a naïve mammal also is encompassed by the scope of theembodiments. The antibody, antibody fragment, antibody-like molecule, ormolecule with a high affinity to a specific tumor marker is used totarget the Peptide conjugate to a specific cellular or tissue target.For example, a tumor with a distinctive surface antigen or expressedantigen can be targeted by the antibody, antibody fragment, orantibody-like binding molecule and the tumor cells can be killed by thePeptide. Such an approach using antibody targeting has the anticipatedadvantages of decreasing dosage, increasing the likelihood of binding toand uptake by the target cells, and increased usefulness for targetingand treating metastatic tumors and microscopic sized tumors.

The embodiments also encompass the use of NTP peptides conjugated orlinked or bound to a protein or other molecule to form a compositionthat, upon cleavage at or near the site(s) of the tumor or otherunwanted cells by a tumor- or site-specific enzyme or protease or by anantibody conjugate that targets tumor or other unwanted cells, releasesthe Peptide at or near the site(s) of the tumor or other unwanted cells

The embodiments also encompass the use of NTP peptides conjugated orlinked or bound to a protein or other molecule to form a compositionthat releases the Peptide or some biologically active fragment of thePeptide upon exposure of the tissue to be treated to light (as in lasertherapies or other photo-dynamic or photo-activated therapy), otherforms of electromagnetic radiation such as infra-red radiation,ultraviolet radiation, x-ray or gamma ray radiation, localized heat,alpha or beta radiation, ultrasonic emissions, or other sources oflocalized energy.

The embodiments also encompass therapeutic compositions of NTP peptidesemploying dendrimers, fullerenes, and other synthetic molecules,polymers and macromolecules where the Peptide and/or its correspondingDNA molecule is conjugated with, attached to or enclosed in themolecule, polymer or macromolecule, either by itself or in conjunctionwith other species of molecule such as a tumor-specific marker. Forexample, U.S. Pat. No. 5,714,166, Bioactive and/or Targeted DendimerConjugates, provides a method of preparing and using, inter alia,dendritic polymer conjugates composed of at least one dendrimer with atarget director(s) and at least one bioactive agent conjugated to it.The disclosure of U.S. Pat. No. 5,714,166 is incorporated by referenceherein in its entirety.

The embodiments also encompasses methods of treating mammals withtherapeutic compositions of NTP peptides and/or genes and drug deliveryvehicles such as lipid emulsions, micelle polymers, polymermicrospheres, electroactive polymers, hydrogels and liposomes, incombination with an additional active agent.

The use of NTP peptides or related genes or gene equivalents transferredto the unwanted cells in a mammal also is encompassed by theembodiments. Overexpression of the NTP peptide within the tumor can beused to induce the cells in the tumor to die and thus reduce the tumorcell population. The gene or gene equivalent transfer of NTP peptide totreat the unwanted cellular elements is anticipated to have theadvantage of requiring less dosage, and of being passed on to thecellular progeny of the targeted cellular elements, thus necessitatingless frequent therapy, and less total therapy. This embodiment alsoencompasses the transfer of genes that code for a fusion proteincontaining an NTP peptide to the unwanted cells or neighboring cellswhere, following the expression of the gene and the production and/orsecretion of the fusion protein, the fusion protein is cleaved either bynative enzymes or proteases or by a prodrug to release the NTP peptidein, at or near the unwanted cells.

The use of cloned recombinant peptide-antibody conjugates; clonedrecombinant peptide-antibody fragment conjugates; and cloned recombinantpeptide-antibody-like protein conjugates for administration to treatmentnaïve mammals also is encompassed by the scope of the embodiments. Theadvantages of a cloned NTP peptide combined with targeting conjugate(such as an antibody, antibody fragment, antibody-like molecule, or amolecule with a high affinity to a cancer-specific receptor or othertumor marker) are that such a molecule combines the targeting advantagesdescribed above in addition to advantages for manufacturing andstandardized production of the cloned conjugated molecule.

The embodiments further include the use of therapeutic compositions ofNTP peptides or genes or gene equivalents as a component of the coatingof a medical device such as a stent in order to remove, inhibit orprevent unwanted cellular proliferation or accumulation, in combinationwith an additional active agent.

Solid dosage forms for oral administration include but are not limitedto, capsules, tablets, pills, powders, and granules. In such soliddosage forms, the additional active agent, and/or the NTP peptide can beadmixed with at least one of the following: (a) one or more inertexcipients (or carrier), such as sodium citrate or dicalcium phosphate;(b) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and silicic acid; (c) binders, such as carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (d)humectants, such as glycerol; (e) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain complex silicates, and sodium carbonate; (f) solution retarders,such as paraffin; (g) absorption accelerators, such as quaternaryammonium compounds; (h) wetting agents, such as acetyl alcohol andglycerol monostearate; (i) adsorbents, such as kaolin and bentonite; and(j) lubricants, such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.For capsules, tablets, and pills, the dosage forms may also comprisebuffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may compriseinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents, and emulsifiers. Exemplary emulsifiersare ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Actual dosage levels of active ingredients in the compositions of theembodiments may be varied to obtain an amount of NTP peptide andadditional active agent that is effective to obtain a desiredtherapeutic response for a particular composition and method ofadministration. The selected dosage level therefore depends upon thedesired therapeutic effect, the route of administration, the desiredduration of treatment, and other factors.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. The interrelationship ofdosages for animals of various sizes, species and humans (based on mg/M²of body surface) is described by E. J. Freireich et al., CancerChemother. Rep., 50 (4):219 (1966). Body surface area may beapproximately determined from the height and weight of an individual(see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp.537-538 (1970)).

The total daily dose of the NTP peptide and additional active agentadministered to a host may be in single or divided doses. Dosage unitcompositions may contain such amounts of such submultiples thereof asmay be used to make up the daily dose. It will be understood, however,that the specific dose level for any particular patient will depend upona variety of factors including the body weight, general health, sex,diet, time and route of administration, potency of the administereddrug, rates of absorption and excretion, combination with other drugsand the severity of the particular disease being treated. It ispreferred that the composition is administered only once as an injectionor infusion, or in another preferred embodiment, the composition isadministered twice. In this embodiment, the period of time betweenadministration of the composition may vary anywhere from 2 months to 10years, or from 8 months to 4 years, or more than about one year (e.g.,between 1 and 2 years).

A method of administering an NTP peptide composition according to theembodiments includes, but is not limited to, administering the compoundsintramuscularly, orally, intravenously, intraperitoneally,intracerebrally (intraparenchymally), intracerebroventricularly,intratumorally, intralesionally, intradermally, intrathecally,intranasally, intraocularly, intraarterially, topically, transdermally,via an aerosol, infusion, bolus injection, implantation device,sustained release system etc.

Another method of administering an NTP peptide of the embodiments is bya transdermal or transcutaneous route. The additional active agent maybe employed together with the NTP peptide, or may be administeredseparately as discussed above, or may not be administered at all. Oneexample of such an embodiment is the use of a patch. In particular, apatch can be prepared with a fine suspension of Peptide in, for example,dimethylsulfoxide (DMSO), or a mixture of DMSO with cottonseed oil andbrought into contact with the skin of the tumor carrying mammals awayfrom the tumor location site inside a skin pouch. Other mediums ormixtures thereof with other solvents and solid supports would workequally as well. The patch can contain the Peptide compound in the formof a solution or a suspension. The patch can then be applied to the skinof the patient, for example, by means of inserting it into a skin pouchof the patient formed by folding and holding the skin together by meansof stitches, clips or other holding devices. This pouch should beemployed in such a manner so that continuous contact with the skin isassured without the interference of the mammal. Besides using a skinpouch, any device can be used which ensures the firm placement of thepatch in contact with the skin. For instance, an adhesive bandage couldbe used to hold the patch in place on the skin.

NTP peptides, optionally in combination with an additional active agent,may be administered in a sustained release formulation or preparation.Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers, 22: 547-556), poly(2-hydroxyethyl-methacrylate) (Langer etal., J. Biomed. Mater. Res., 15: 167-277 and Langer, Chem. Tech., 12:98-105), ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also may include liposomes, which can be prepared by any ofseveral methods known in the art (e.g., Eppstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692; EP 36,676; EP 88,046; and EP 143,949).

Another method of administering an NTP peptide of the embodiments is bydirect or indirect infusion of the NTP peptide into the tumor or othertissue to be treated. One example of such an embodiment is the directinjection of NTP peptide into the tumor or other tissue to be treated.The treatment may consist of a single injection, multiple injections onone occasion or a series of injections over a period of hours, days ormonths with the regression or destruction of the tumor or other tissueto be treated being monitored by means of biopsy, imaging or othermethods of monitoring tissue growth. The injection into the tumor orother tissue to be treated may be by a device inserted into an orificesuch as the nose, mouth, ear, vagina, rectum or urethra or through anincision in order to reach the tumor or tissue in vivo and may performedin conjunction with an imaging or optical system such as ultrasound orfibre optic scope in order to identify the appropriate site for theinjection(s). Another example of such an embodiment is the use of adevice that can provide a constant infusion of NTP peptide to the tissueover time.

Another method of administering an NTP peptide is in conjunction with asurgical or similar procedure employed to physically excise, ablate orotherwise kill or destroy tumor or other tissue or cellular elementsrequired or desired to be removed or destroyed wherein an NTP peptide ofthe embodiments is administered to the immediate area(s) surrounding thearea(s) where the tumor or other tissue was removed in order to destroyor impede the growth of any tumor cells or other cellular elements notremoved or destroyed by the procedure

Another method of administering an NTP peptide of the embodiments is byimplantation of a device within the tumor or other tissue to be treated.In this embodiment, the additional active agent typically will beadministered via a different route of administration than the NTPpeptide. One example of such an embodiment is the implantation of awafer containing Peptide in the tumor or other tissue to be treated, andthe administration of the additional active agent via oraladministration. The wafer releases a therapeutic dose of NTP peptideinto the tissue over time. Alternatively or additionally, thecomposition may be administered locally via implantation into theaffected area of a membrane, sponge, or other appropriate material on towhich the NTP peptide has been absorbed. Where an implantation device isused, the device may be implanted into any suitable tissue or organ, anddelivery of the Peptide may be directly through the device via bolus, orvia continuous administration, or via catheter using continuousinfusion.

An alternative method of administration is to introduce one or morecopies of an NTP peptide-encoding gene into the cell being targeted and,if necessary, inducing the copy(ies) of the gene to begin producingPeptide intracellularly. In this embodiment, the additional active agenttypically will be administered via a different route of administrationthan the NTP peptide. One manner in which gene therapy can be applied isto use the NTP peptide-encoding gene (either genomic DNA, cDNA, and/orsynthetic DNA encoding the Peptide (or a fragment, variant, homologue orderivative thereof)) which may be operably linked to a constitutive orinducible promoter to form a gene therapy DNA construct. The promotermay be homologous or heterologous to an endogenous Peptide-encodinggene, provided that it is active in the cell or tissue type into whichthe construct will be inserted. Other components of the gene therapy DNAconstruct may optionally include, as required, DNA molecules designedfor site-specific integration (e.g., endogenous flanking sequencesuseful for homologous recombination), tissue-specific promoter,enhancer(s) or silencer(s), DNA molecules capable of providing aselective advantage over the parent cell, DNA molecules useful as labelsto identify transformed cells, negative selection systems, cell specificbinding agents (as, for example, for cell targeting) cell-specificinternalization factors, and transcription factors to enhance expressionby a vector as well as factors to enable vector manufacture.

Means of gene delivery to a cell or tissue in vivo or ex vivo include(but are not limited to) direct injection of bare DNA, ballisticmethods, liposome-mediated transfer, receptor-mediated transfer(ligand-DNA complex), electroporation, and calcium phosphateprecipitation. See U.S. Pat. No. 4,970,154, WO 96/40958, U.S. Pat. No.5,679,559, U.S. Pat. No. 5,676,954, and U.S. Pat. No. 5,593,875, thedisclosures of each of which are incorporated by reference herein intheir entirety. They also include use of a viral vector such as aretrovirus, adenovirus, adeno-associated virus, pox virus, lentivirus,papilloma virus or herpes simplex virus, use of a DNA-protein conjugateand use of a liposome. The use of gene therapy vectors is described, forexample, in U.S. Pat. No. 5,672,344, U.S. Pat. No. 5,399,346, U.S. Pat.No. 5,631,236, and U.S. Pat. No. 5,635,399, the disclosures of each ofwhich are incorporated by reference herein in their entirety.

The NTP peptide-encoding gene may be delivered through implanting intopatients certain cells that have been genetically engineered ex vivo,using methods such as those described herein, to express and secrete theNTP peptide or fragments, variants, homologues, or derivatives thereof.Such cells may be animal or human cells, and may be derived from thepatient's own tissue or from another source, either human or non-human.Optionally, the cells may be immortalized or be stem cells. However, inorder to decrease the chance of an immunological response, it ispreferred that the cells be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow release of the protein product(s) but prevent destruction of thecells by the patient's immune system or by other detrimental factorsfrom the surrounding tissues. Methods used for membrane encapsulation ofcells are familiar to the skilled artisan, and preparation ofencapsulated cells and their implantation in patients may beaccomplished without undue experimentation. See, e.g., U.S. Pat. Nos.4,892,538; 5,011,472; and 5,106,627, the disclosures of each of whichare incorporated by reference herein in their entirety. A system forencapsulating living cells is described in PCT WO 91/10425. Techniquesfor formulating a variety of other sustained or controlled deliverymeans, such as liposome carriers, bio-erodible particles or beads, arealso known to those in the art, and are described, for example, in U.S.Pat. No. 5,653,975, the disclosure of which is incorporated by referenceherein in their entirety. The cells, with or without encapsulation, maybe implanted into suitable body tissues or organs of the patient.

In certain embodiments, the invention provides a method of reducing theincidence of clinically detected prostate cancer in mammals, includingmammals suffering from BPH, which comprises administering to the mammalat least once, a therapeutically effective amount of an NTP peptide,specifically an isolated peptide comprising the amino acid sequence inSEQ ID NO. 66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).In other embodiments, the invention provides a method of preventing orreducing the progression of prostate cancer in mammals at risk ofdeveloping clinically detected prostate cancer, which comprisesadministering to the mammal a therapeutically effective amount of an NTPpeptide, specifically an isolated peptide comprising the amino acidsequence in SEQ ID NO. 66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).

In certain embodiments, the isolated peptide comprising the amino acidsequence in SEQ ID NO. 66 is administered in combination with at leastone active agent selected from the group consisting of (1) of aninhibitor of 5α-reductase and/or an antiestrogen, (2) an inhibitor of5α-reductase and/or an aromatase inhibitor, (3) a 5α-reductase inhibitorand/or a 17β-HSD inhibitor, (4) a 5α-reductase inhibitor, anantiestrogen and an aromatase inhibitor, (5) a 5α-reductase inhibitor,an antiestrogen and a 17β-HSD inhibitor, (6) a 5α-reductase inhibitor,an aromatase inhibitor, an antiestrogen and a 17β-HSD inhibitor, (7) a5α-reductase inhibitor, an antiandrogen and an antiestrogen, (8), a5α-reductase inhibitor, an antiandrogen and an aromatase inhibitor, (9)a 5α-reductase inhibitor, an antiandrogen and an 17β-HSD inhibitor, (10)a 5α-reductase inhibitor, an antiandrogen, an antiestrogen and anaromatase inhibitor, (11) a 5α-reductase inhibitor, an antiandrogen, anaromatase inhibitor and a 17β-HSD inhibitor, (12) a 5α-reductaseinhibitor, an antiandrogen, an aromatase inhibitor, an antiestrogen anda 17β-HSD inhibitor, (13) a 17β-HSD inhibitor and an antiestrogen, (14)a 17β-HSD inhibitor and an aromatase inhibitor, (15) a 17β-HSDinhibitor, an aromatase inhibitor and an antiestrogen, (16) a 17β-HSDinhibitor, an antiandrogen and an antiestrogen, (17) a 17β-HSDinhibitor, an antiandrogen and an aromatase inhibitor, (18) a 17β-HSDinhibitor, an antiandrogen, an antiestrogen and an aromatase inhibitor,(19) an antiestrogen and an aromatase inhibitor and (20) anantiestrogen, an aromatase inhibitor, and an antiandrogen, (21) an LHRHagonist or antagonist, an inhibitor of 5α-reductase and an antiestrogen,(22) an LHRH agonist or antagonist, an inhibitor of 5α-reductase and anaromatase inhibitor, (23) an LHRH agonist or antagonist, a 5α reductaseinhibitor and a 17β-HSD inhibitor, (24) an LHRH agonist or antagonist, a5α-reductase inhibitor, an antiestrogen and an aromatase inhibitor, (25)an LHRH agonist or antagonist, a 5α-reductase inhibitor, an antiestrogenand a 17β-HSD inhibitor, (26) an LHRH agonist or antagonist, a5α-reductase inhibitor, an aromatase inhibitor, an antiestrogen and a17β-HSD inhibitor, (27) an LHRH agonist or antagonist, a 5α-reductaseinhibitor, an antiandrogen and an antiestrogen, (28), an LHRH agonist orantagonist, a 5α-reductase inhibitor, an antiandrogen and an aromataseinhibitor, (29) an LHRH agonist or antagonist, a 5α-reductase inhibitor,an antiandrogen and an 17β-HSD inhibitor, (30) an LHRH agonist orantagonist, a 5α-reductase inhibitor, an antiandrogen, an antiestrogenand an aromatase inhibitor, (31) an LHRH agonist or antagonist, a5α-reductase inhibitor, an antiandrogen, an aromatase inhibitor and a17β-HSD inhibitor, (32) an LHRH agonist or antagonist, a 5α-reductaseinhibitor, an antiandrogen, an aromatase inhibitor, an antiestrogen anda 17β-HSD inhibitor, (33) an LHRH agonist or antagonist, a 17β-HSDinhibitor and an antiestrogen, (34) an LHRH agonist or antagonist, a17β-HSD inhibitor and an aromatase inhibitor, (35) an LHRH agonist orantagonist, a 17β-HSD inhibitor, an aromatase inhibitor and anantiestrogen, (36) an LHRH agonist or antagonist, a 17β-HSD inhibitor,an antiandrogen and an antiestrogen, (37) an LHRH agonist or antagonist,a 17β-HSD inhibitor, an antiandrogen and an aromatase inhibitor, (38) anLHRH agonist or antagonist, a 17β-HSD inhibitor, an antiandrogen, anantiestrogen and an aromatase inhibitor, (39) an LHRH agonist orantagonist, an antiestrogen and an aromatase inhibitor and (40) an LHRHagonist or antagonist, an antiestrogen, an aromatase inhibitor, and anantiandrogen.

The following examples are provided to illustrate the presentembodiments. It should be understood, however, that the embodiments arenot to be limited to the specific conditions or details described inthese examples. Throughout the specification, any and all references toa publicly available document, including a U.S. patent, are specificallyincorporated by reference. In particular, the embodiments expresslyincorporate by reference the examples contained in pending U.S. patentapplication Ser. No. 14/808,713, filed Jul. 24, 2015, entitled: METHODSOF REDUCING THE NEED FOR SURGERY IN PATIENTS SUFFERING FROM BENIGNPROSTATIC HYPERPLASIA; U.S. patent application Ser. No. 14/606,683,filed Jan. 27, 2015, entitled: METHOD OF TREATING DISORDERS REQUIRINGDESTRUCTION OR REMOVAL OF CELLS, U.S. application Ser. No. 14/738,551,filed Jun. 12, 2015, entitled: COMBINATION COMPOSITIONS FOR TREATINGDISORDERS REQUIRING REMOVAL OR DESTRUCTION OF UNWANTED CELLULARPROLIFERATIONS, U.S. patent application Publication Nos. 2007/0237780(now abandoned); 2003/0054990 (now U.S. Pat. No. 7,172,893);2003/0096350 (now U.S. Pat. No. 6,924,266); 2003/0096756 (now U.S. Pat.No. 7,192,929); 2003/0109437 (now U.S. Pat. No. 7,241,738); 2003/0166569(now U.S. Pat. No. 7,317,077); 2005/0032704 (now U.S. Pat. No.7,408,021); and 2015/0148303 (now U.S. Pat. No. 9,243,035), each ofwhich reveal that certain peptides specified therein are effectiveagents for causing cell death in vivo in normal rodent muscle tissue,subcutaneous connective tissue, dermis and other tissue.

Example One

In a series of clinical studies undertaken between 2009 and 2016 a totalof 628 men with benign prostatic hyperplasia (BPH) received SEQ ID NO.66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),2.5 mg given by intraprostatic injection into the transition zone of theprostate to treat the symptoms of BPH and were subsequently followedclinically and by laboratory testing. After intervals up to 6.5 yearsafter injection, all available patients were followed urologically atspecialized clinics with investigation of BPH symptoms and events ofdisease progression and safety assessments. All cases of clinically orbiopsy detected prostate cancer were documented. The incidence of newbiopsy or surgically proven prostate cancer after 12 months and overallwas compared to the incidence of detected prostate cancer in largelong-term clinical trials in the medical literature. Although a) thetreatments were for BPH; b) the injections were directed at the centralpart of the prostate where cancer is less common; and c) the injectionsconsisted of only a single or two injection(s) of SEQ ID NO. 66, theincidence of prostate cancer compared to the known expected incidence of2-25% was surprisingly reduced in these patients to 0-1.3%. The resultscan be found in Table 1 below.

TABLE 1 Treatment Number of patients Incidence of cancer SEQ ID NO: 66¹628   0.8% Control²  >20% ¹Excludes patients diagnosed with prostatecancer in first 12 months of study. Incidence of clinically detectedcancer from inclusion of patients diagnosed with prostate cancer infirst 12 months of study was 1.3%; ²Long term trial outcome results inBPH populations from published studies. (Thompson, IM, et al., “TheInfluence of Finasteride on the Development of Prostate Cancer,” N EnglJ Med, Vol.349, pp. 215-224 (2003); Andriole GL, et al., “Effect ofDutasteride on the Risk of Prostate Cancer,” N Engl J Med, Vol. 362, pp.1192-1202 (2010)).

Based on the results of these studies, administering the NTP peptides ofthe present invention to patients requiring treatment for, orsusceptible to developing, prostate cancer, and/or administration toportions of the prostate where cancer is more common, would be expectedto have an even more pronounced effect in preventing the incidence ofprostate cancer, as well as preventing or reducing the progression ofprostate cancer in patients susceptible to developing prostate cancer asdetermined by one or more prostate cancer screening techniques. Theclaimed compositions therefore are useful in treating a patientpopulation different from only those suffering from BPH, and are usefulin preventing the incidence of prostate cancer, as well as preventing orreducing the progression of prostate cancer in patients susceptible todeveloping prostate cancer as determined by one or more prostate cancerscreening techniques for this patient population.

Example Two

In a series of clinical studies undertaken between 2009 and 2016, atotal of 816 men with benign prostatic hyperplasia (BPH) received SEQ IDNO. 66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),2.5 mg (n=628) or vehicle alone injections of sterile saline solution(n=188) given by intraprostatic injection into the transition zone ofthe prostate to treat the symptoms of BPH, were subsequently followedclinically and by laboratory testing. After intervals up to 6.5 yearsafter injection, all available patients were followed urologically atspecialized clinics with investigation of BPH symptoms and events ofdisease progression and safety assessments. All cases of clinically orbiopsy detected prostate cancer were documented. The incidence of newbiopsy or surgically proven prostate cancer after 12 months and overallwas compared to the incidence of detected prostate cancer in thepatients receiving the sterile saline solution (control). Although a)the treatments were for BPH; b) the injections were directed at thecentral part of the prostate where cancer is less common; and c) theinjections consisted of only a single or two injection(s) of SEQ ID NO.66, the incidence of prostate cancer in the subjects treated inaccordance with the present invention was unexpectedly much lower thanthat of the control population. The results are shown in Table 2 below.

TABLE 2 Treatment Number of patients Incidence of cancer¹ SEQ ID NO: 66²628 0.8%* Control¹ 188 2.7%  ¹Excludes patients diagnosed with prostatecancer in first 12 months of study. ²Incidence of clinically detectedcancer from inclusion of patients diagnosed with prostate cancer infirst 12 months of study was 1.3%; *p < .05

Based on the results of these studies, administering the NTP peptides ofthe present invention to patients requiring treatment for, orsusceptible to developing, prostate cancer, and/or administration toportions of the prostate where cancer is more common, would be expectedto have an even more pronounced effect in preventing the incidence ofprostate cancer, as well as preventing or reducing the progression ofprostate cancer in patients susceptible to developing prostate cancer asdetermined by one or more prostate cancer screening techniques. Theclaimed compositions therefore are useful in treating a patientpopulation different from only those suffering from BPH, and are usefulin preventing the incidence of prostate cancer, as well as preventing orreducing the progression of prostate cancer in patients susceptible todeveloping prostate cancer as determined by one or more prostate cancerscreening techniques for this patient population.

Example Three

In a series of clinical studies undertaken between 2009 and 2016 a totalof 628 men with benign prostatic hyperplasia (BPH) received SEQ ID NO.66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),2.5 mg given by intraprostatic injection into the transition zone of theprostate to treat the symptoms of BPH and were subsequently followedclinically and by laboratory testing. After intervals up to 6.5 yearsafter injection, all available patients were followed urologically atspecialized clinics with investigation of BPH symptoms and events ofdisease progression and safety assessments. All cases of clinically orbiopsy detected prostate cancer were documented. The incidence of newbiopsy or surgically proven prostate cancer of Gleason Grade>=7 wascompared to the incidence of detected prostate cancer in large long-termclinical trials in the medical literature. Although a) the treatmentswere for BPH; b) the injections were directed at the central part of theprostate where cancer is less common; and c) the injections consisted ofonly a single or two injection(s) of SEQ ID NO. 66, the incidence ofprostate cancer compared to the known expected incidence of 5-6% wassurprisingly reduced in these patients to <0.3%. The results can befound in Table 3 below.

TABLE 3 Number Incidence of cancer Treatment of patients (GleasonGrade >= 7) SEQ ID NO: 66 628 <0.3% Control¹   5-6% ¹Long term trialoutcome results in BPH populations from published studies. (Thompson,IM, et al., “The Influence of Finasteride on the Development of ProstateCancer,” N Engl J Med, Vol.349, pp. 215-224 (2003); Andriole GL, et al.,“Effect of Dutasteride on the Risk of Prostate Cancer,” N Engl J Med,Vol. 362, pp. 1192-1202 (2010)).

Based on the results of these studies, administering the NTP peptides ofthe present invention to patients requiring treatment for, orsusceptible to developing, prostate cancer, and/or administration toportions of the prostate where cancer is more common, would be expectedto have an even more pronounced effect in preventing the incidence ofprostate cancer, as well as preventing or reducing the progression ofprostate cancer in patients susceptible to developing prostate cancer asdetermined by one or more prostate cancer screening techniques. Theclaimed compositions therefore are useful in treating a patientpopulation different from only those suffering from BPH, and are usefulin preventing the incidence of prostate cancer, as well as preventing orreducing the progression of prostate cancer in patients susceptible todeveloping prostate cancer as determined by one or more prostate cancerscreening techniques for this patient population.

The results from the foregoing examples illustrate the unexpectedlysuperior effect of the NTP peptides, in reducing the incidence ofclinically detected prostate cancer, and in preventing or reducing theprogression of prostate cancer in patients susceptible to developingprostate cancer as determined by one or more prostate cancer screeningtechniques. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present embodiments without departing from the spirit or scope ofthe embodiments.

What is claimed is:
 1. A method of reducing the onset of prostate cancerin a mammal susceptible to developing prostate cancer as determined byone or more prostate cancer screening techniques, comprisingadministering, more than once, to the mammal susceptible to developingprostate cancer a therapeutically effective amount of an isolatedpeptide comprising the amino acid sequence in SEQ ID NO. 66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),wherein the method reduces the onset of prostate cancer by an amountgreater than about 15%, when compared to mammals receiving a placebo. 2.The method of claim 1, wherein the method comprises administration of atherapeutically effective amount of at least one of the peptides asclaimed in claim 1 and a carrier.
 3. The method of claim 1, wherein themethod comprises administration of a therapeutically effective amount ofat least one of the peptides as claimed in claim 1 and at least one andup to 25 additional amino acids flanking either the N-terminus orC-terminus of the isolated peptide of claim
 1. 4. The method of claim 1,wherein the peptide is administered by a method selected from the groupconsisting of orally, subcutaneously, intradermally, intranasally,intravenously, intramuscularly, intrathecally, intranasally,intratumorally, topically, and transdermally.
 5. The method of claim 1,wherein the method provides an onset of prostate cancer in a range offrom about 0% to about 5%.
 6. The method of claim 5, wherein the methodprovides an onset of prostate cancer in a range of from about 0% toabout 2%.
 7. The method of claim 5, wherein the method provides an onsetof prostate cancer in a range of from about 0% to about 1.3%.
 8. Amethod of reducing the progression of prostate cancer in a mammalsusceptible to developing prostate cancer as determined by one or moreprostate cancer screening techniques, comprising administering, morethan once, to the mammal a therapeutically effective amount of anisolated peptide comprising the amino acid sequence in SEQ ID NO. 66(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu),wherein the method reduces the progression of prostate cancer to anamount within the range of from about 0% to less than about 5%.
 9. Themethod of claim 8, wherein the method comprises administration of atherapeutically effective amount of at least one of the peptides asclaimed in claim 8, and a carrier.
 10. The method of claim 8, whereinthe method comprises administration of a therapeutically effectiveamount of at least one of the peptides as claimed in claim 8 and atleast one and up to 25 additional amino acids flanking either theN-terminus or C-terminus of the isolated peptide of claim
 8. 11. Themethod of claim 8, wherein the peptide is administered by a methodselected from the group consisting of orally, subcutaneously,intradermally, intranasally, intravenously, intraprostatically,intramuscularly, intrathecally, intranasally, intratumorally, topically,and transdermally.
 12. The method of claim 8, wherein the method reducesthe progression of prostate cancer to an amount within the range about0% to about 1%.
 13. The method of claim 8, wherein the method reducesthe progression of prostate cancer to an amount of about 0%.
 14. Themethod of claim 8, wherein the mammal susceptible to developing prostatecancer had a baseline PSA level of ≥2.5.